Patent application title: ANTI-FIBROTIC COMPOSITIONS COMPRISING FENDRR OR FRAGMENTS OR VARIANTS THEREOF AND METHODS OF PRODUCTION AND USE THEREOF
Inventors:
IPC8 Class: AC12N15113FI
USPC Class:
1 1
Class name:
Publication date: 2021-04-29
Patent application number: 20210123062
Abstract:
Compositions containing long non-coding RNAs (IncRNA's) or fragments or
variants thereof are disclosed. Also disclosed are compositions
containing vectors that include or encode the IncRNA's or
fragments/variants thereof. Further disclosed are methods of producing
and using these compositions containing or encoding the IncRNA's.Claims:
1. An anti-fibrotic composition, comprising: an isolated and/or purified
Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a
fragment or variant thereof; and a pharmaceutically-acceptable carrier.
2. The anti-fibrotic composition of claim 1, wherein the FENDRR IncRNA or fragment or variant thereof is encoded by a sequence comprising at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
3. The anti-fibrotic composition of claim 1, wherein the FENDRR IncRNA or fragment or variant thereof is encapsulated.
4. The anti-fibrotic composition of claim 3, further comprising a delivery vehicle in which the FENDRR IncRNA or fragment or variant thereof is encapsulated, wherein the delivery vehicle is selected from the group consisting of a liposome, a lipoplex, a microvesicle, an exosome, a lipidoid nanoparticle, a polymeric nanoparticle, an inorganic nanoparticle, and a stable nucleic acid particle (SNALP).
5. A pharmaceutical composition, comprising: a vector comprising a sequence encoding at least about 100 contiguous nucleotides of a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof; and a pharmaceutically-acceptable carrier.
6. The pharmaceutical composition of claim 5, wherein the sequence comprises at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
7. The pharmaceutical composition of claim 5, wherein the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, an alpha viral vector, a herpes viral vector, a lentiviral vector, a measles viral vector, a pox viral vector, a phage vector, or a retroviral vector.
8. The pharmaceutical composition of claim 5, wherein the vector further comprises an expression control sequence to which the sequence is operably linked.
9. A method of inhibiting activation of lung fibroblasts, the method comprising the step of: contacting the lung fibroblasts with a composition selected from the group consisting of: (i) an isolated and/or purified Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof; or (ii) a vector comprising a sequence encoding at least about 100 contiguous nucleotides of a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof.
10. The method of claim 9, wherein in (i), the FENDRR IncRNA or fragment or variant thereof is encoded by a sequence comprising at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids, and wherein in (ii), the sequence comprises at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
11. The method of claim 9, wherein in (i), the FENDRR IncRNA or fragment or variant thereof is encapsulated in a delivery vehicle, wherein the delivery vehicle is selected from the group consisting of a liposome, lipid nanoparticles, a lipoplex, a microvesicle, an exosome, a lipidoid nanoparticle, a polymeric nanoparticle, an inorganic nanoparticle, and a stable nucleic acid particle (SNALP).
12. The method of claim 9, wherein in (ii), the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, an alpha viral vector, a herpes viral vector, a lentiviral vector, a measles viral vector, a pox viral vector, a phage vector, or a retroviral vector.
13. The method of claim 9, wherein in (ii), the vector further comprises an expression control sequence to which the sequence is operably linked.
14. A method of treating or reducing the occurrence of pulmonary fibrosis in a subject, comprising the step of: administering a composition to the subject, wherein the composition is selected from the group consisting of: (i) a composition comprising a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof and a pharmaceutically-acceptable carrier; or (ii) a composition comprising a vector and a pharmaceutically-acceptable carrier, wherein the vector comprises a sequence encoding at least about 100 contiguous nucleotides of a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof.
15. The method of claim 14, wherein the composition is administered via a route selected from the group consisting of oral, topical, transdermal, parenteral, subcutaneous, intranasal, intratracheal, intrabronchial, mucosal, intramuscular, intraperitoneal, intravitreal, and intravenous routes.
16. The method of claim 14, wherein in (i), the FENDRR IncRNA or fragment or variant thereof is encoded by a sequence comprising at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids, and wherein in (ii), the sequence comprises at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
17. The method of claim 14, wherein in (i), the FENDRR IncRNA or fragment or variant thereof is encapsulated in a delivery vehicle, wherein the delivery vehicle is selected from the group consisting of a liposome, a lipoplex, a microvesicle, an exosome, a lipidoid nanoparticle, a polymeric nanoparticle, an inorganic nanoparticle, and a stable nucleic acid particle (SNALP).
18. The method of claim 14, wherein in (ii), the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, an alpha viral vector, a herpes viral vector, a lentiviral vector, a measles viral vector, a pox viral vector, a phage vector, or a retroviral vector.
19. The method of claim 14, wherein in (ii), the vector further comprises an expression control sequence to which the sequence is operably linked.
20. The method of claim 14, further defined as a method of treating or reducing the occurrence of idiopathic pulmonary fibrosis in a subject.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE
STATEMENT
[0001] The subject application claims benefit under 35 USC .sctn. 119(e) of provisional application U.S. Ser. No. 62/926,797, filed Oct. 28, 2019. The entire contents of the above-referenced patent(s)/patent application(s) are hereby expressly incorporated herein by reference.
BACKGROUND
[0003] Idiopathic Pulmonary Fibrosis (IPF) is a chronic and lethal fibrotic lung disease characterized by scarring of lung tissues and worsening lung function. Historically, lung tissues from IPF patients show similar characteristics as usual interstitial pneumonia (UIP). The disease primarily occurs in individuals between the ages of 50 and 70 and more frequently occurs in men. The course of IPF is difficult to predict, with a median survival time of 3 to 5 years after diagnosis; there is currently no effective therapy.
[0004] The human genome generates more non-coding RNAs (ncRNAs) than protein-coding RNA sequences. Long non-coding RNAs (IncRNAs) are ncRNAs that are typically longer than 200 nt and are transcribed from DNA that was once thought to be "junk." These RNAs are larger than small ncRNAs such as microRNAs, Piwi-interacting RNAs, siRNAs, and small nucleolar RNAs, which are typically about 20-180 nt in length. Current estimates suggest that about 20,000 distinct IncRNAs are present in humans.
[0005] Most ncRNAs, including long non-coding RNAs (IncRNAs), are synthesized by polymerase II. IncRNAs are mRNA-like transcripts, but are non-protein encoding RNA molecules that are more than 200 bp long. IncRNAs are processed by capping, splicing, and polyadenylation, which is similar to the process of protein-coding genes. Only a small number of IncRNAs have been annotated functionally. IncRNAs are located in the nucleus, cytoplasm, and mitochondria, where they participate in various molecular functions, such as chromatin remodeling, transcriptional regulation, RNA splicing, RNA stability, and translation control. IncRNAs are important in controlling critical physiological functions, including gene imprinting, cell proliferation, differentiation, apoptosis, autophagy, migration, immune responses, and chromosome structure. Aberrant expression of IncRNAs has been associated with a broad range of human diseases, including cardiovascular, neurodegenerative, metabolic, and lung diseases, as well as tumors and infections.
[0006] Fetal-lethal noncoding developmental regulatory RNA (FENDRR) is a IncRNA that is transcribed bidirectionally with FOXF1 on its opposite strand. FENDRR binds to polycomb repressive complex 2 (PRC2) and/or TrxG/MLL complexes to epigenetically regulate the expression of its target genes. Murine Fendrr is essential for normal development of the lungs, heart, and body wall. LacZ reporter profiling has shown that Fendrr is highly expressed in embryonic and adult lung tissue. Fendrr homozygotes are embryonic-lethal due to defective structural maturation of the lungs. Genomic deletion within the FENDRR gene was found in a human fatal lung development disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV). FENDRR has been linked to other human diseases, such as gastric cancer.
[0007] Therefore, there is a need in the art for new and improved compositions that contain or encode IncRNAs, such as (but not limited to), FENDRR. There is also a need in the art for methods of inhibiting activation of fibroblasts and treating or reducing the occurrence of pulmonary fibrosis. It is to such compositions and methods that the present disclosure is directed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0009] FIG. 1. FENDRR was down-regulated in fibrotic lungs and fibroblasts. Panel (A): Next-generation RNA sequencing analysis showing the down-regulation of FENDRR in IPF lungs in two published datasets. Panel (B): Real-time PCR showing FENDRR down-regulation in LTRC IPF lungs. n=7 for Control, n=27 for IPF. The primers detecting human FENDRR transcript variant 2 and 3 were used. Panel (C): Real-time PCR showing FENDRR down-regulation in fibrotic mouse lungs (n=3). Panel (D): Real-time PCR showing FENDRR expression in primary fibroblasts and alveolar epithelial type I and type II cells (AEC I and AEC II) isolated from saline (Sal)- and bleomycin (Bleo)-treated mice (n=3). The expression levels were normalized to GAPDH. Data are expressed as the percent of the fibroblast control group. The primers detecting mouse Fendrr transcript variants 1 and 2 were used for C and D. Panel (E) shows the copy number of human FENDRR transcript variants in the fibroblasts, as determined by absolute quantitative Real-time PCR (n=3). Panel (F) shows the copy number of human FENDRR variants in LTRC IPF lungs, as determined by droplet digital PCR. n=7 for Control, n=27 for IPF. Panel (G) shows the copy number of mouse Fendrr variants in the lungs of control and bleomycin-treated mice, as determined by droplet digital PCR (n=3). The results are presented as the means.+-.SEM. Student's t-test for A, B, and C and ANOVA, followed by Tukey's HSD test for D, F, and G.
[0010] FIG. 2. Gene structure of FENDRR and primer locations used for determining the expression of FENDRR transcript variants. red color: forward primer binding site; and green color: reverse primer binding site. purple color: common primers for human variants 2, 3 and mouse variants 1, 2.
[0011] FIG. 3. TGF.beta.1-SMAD3 signaling inhibits FENDRR expression in lung fibroblasts. Panel (A): Real-time PCR showing FENDRR down-regulation in HPF fibroblasts treated with TGF.beta.1 (5 ng/ml) for 48 hours (n=3). Panel (B) shows the copy number of FENDRR variants in the control and TGF.beta.1-treated HPF fibroblasts, as determined by droplet digital PCR (n=4). Panel (C): Dual luciferase reporter assay showing the inhibition of human FENDRR promoter activity by TGF.beta.1 in HPF and HFL1 cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Fold change was calculated relative to control (n=3). Panel (D): Western blot analysis showing knockdown of SMAD2/3 expression in LL29 cells by shRNA. Panel (E): Real-time PCR showing that FENDRR expression in LL29 cells was inhibited by TGF.beta.1 and rescued by SMAD3 knockdown (n=3). The results are presented as the means.+-.SEM. Student's t-test for A and ANOVA, followed by Tukey's HSD test for B, C and E.
[0012] FIG. 4. Overexpression of FENDRR inhibits TGF.beta.1-induced fibroblast activation. LL29 cells stably expressing FENDRR transcript variant 3 or a control (VC) were treated with 5 ng/ml of TGF.beta.1 for 48 hours. Panel (A) shows FENDRR expression (n=3). Panel (B) shows that FENDRR overexpression suppressed TGF-.beta.1-induced mRNA expression of .alpha.-SMA, COL1A1, and COL3A1. FENDRR and mRNA expression were determined by real-time PCR and normalized to GAPDH (n=3). Panel (C) shows Western blot analysis demonstrating the suppression of TGF.beta.1-induced .alpha.-SMA, COL1A1, and COL3A1 protein expression levels by FENDRR overexpression. Panel (D) shows FENDRR reduced TGF.beta.1-induced collagen gel contraction. n=4. Panel (E) shows immunostaining demontrating the inhibition of fiber formation by FENDRR using anti-.alpha.-SMA antibodies and Alexa 546-conjugated second antibodies. Scale bar: 50 .mu.m. Panels (F, G): HPF fibroblasts were infected with a lentiviral FENDRR shRNA or its control (shCON) (MOI=50) for two days, and the cells were collected for analysis. Real-time PCR and western blotting showing that the knockdown of FENDRR enhanced mRNA expression of .alpha.-SMA, COL1A1, and COL3A1 and protein expression of .alpha.-SMA and COL1A1. The results are expressed as fold changes relative to shCON. n=3. The results are presented as the means.+-.SEM. Student's t-test for A, F, ANOVA, followed by Tukey's HSD test for B and ANOVA, followed by uncorrected Fisher's LSD test for D.
[0013] FIG. 5. IRP1 is an interacting partner of FENDRR. Panel (A) shows that FENDRR is preferentially localized in the cytoplasm in fibroblasts. The RNA levels in cytoplasmic and nuclear fractions of LL29 and HPF fibroblasts were determined by real-time PCR and calculated with the equation 2.sup.-Ct. GAPDH, ACTB and U2snRNA were used as controls for cytoplasmic and nuclear RNA, respectively (n=3). Panel (B) shows a RIP assay demonstrating the interaction of FENDRR and IRP1 in LL29 fibroblasts (n=3). Panel (C) shows mapping of the binding region of FENDRR with IRP1 by CLIP-qPCR analysis (n=3). Data are presented as the means.+-.SEM. Student's t-test for B.
[0014] FIG. 6. Predicted RNA structure of FENDRR transcript variant 3. Black color: start and ending position. Red color: start and end positions of the IRP1 binding region.
[0015] FIG. 7. FENDRR controls iron metabolism by interacting with IRP1. Panel (A) shows that FENDRR overexpression decreased iron levels in LL29 fibroblasts, as determined with an Iron Assay Kit. n=4. Panel (B) shows that FENDRR overexpression inhibited aconitase activity in LL29 fibroblasts, as measured using an Aconitase Enzyme Activity Microplate Assay Kit. n=3. Panel (C) shows real-time PCR demonstrating the suppression of the expression of TFRC mRNA with FENDRR overexpression. n=4. Panel (D) shows that iron levels were increased in primary fibroblasts isolated from the lungs of bleomycin (Bleo)-treated mice compared to that from saline (Sal)-treated mice (n=3). Panel (E): LL29 cells stably expressing FENDRR or a control (VC) were infected with pooled three lentiviral IRP1 shRNAs or a control (shCON) (MOI=50) for two days, and the cells were then cultured in the serum-free RPMI-1640 medium (iron-free medium) or the complete RPMI-1640 medium containing 10 .mu.M ferric ammonium citrate (iron-supplemented medium) for another two days. Western blotting shows the knockdown of IRP1 expression. IRP1 knockdown rescued the FENDRR-mediated decrease in cellular iron levels in iron-free medium (n=6). Fold changes were calculated based on the VC and shCON control in the iron-free medium. Panels (F, G, H): LL29 lung fibroblasts were treated with 5 .mu.M desferrioxamine (DFO) and with or without TGF.beta.1 (5 ng/ml) for 48 hours. The mRNA expression levels of .alpha.-SMA, COL1A1 and COL3A1 were determined by real-time PCR (n=3). Data are presented as the means.+-.SEM. Student's t-test for A, B, C and D, ANOVA, followed by uncorrected Fisher's LSD test for E, and ANOVA, followed by Tukey's HSD for F, G, and H.
[0016] FIG. 8. miR-214 binding sites in human FENDRR transcript variant 3.
[0017] FIG. 9. FENDRR sponges miR-214. Panel (A) shows a luciferase assay showing that FENDRR overexpression increased the activity of miR-214 sensor in LL29 cells (n=4). Panel (B) shows a luciferase assay showing that FENDRR increased miR-214 sensor activity by competing with miR-214 in HEK293T cells. The effects of FENDRR on a miR-214 sensor were reversed by increasing the miR-214 level. HEK293T cells were co-transfected with 5 ng miR-214 sensor and 20, 40, or 60 ng miR-214/Control and 150 ng FENDRR-overexpression vector/control. Luciferase activities were determined 48 hours after transfection (n=4). Panels (C, D) show Real-time PCR and western blot analysis demonstrating increases in TGF-.beta.1-induced COL1A1 mRNA and protein levels by miR-214 overexpression. LL29 cells were treated with a lentiviral miR-214 or the virus control (VC) at an MOI of 50 for 48 hours. Then, the cells were stimulated with 5 ng/ml of TGF-.beta.1 for 48 hours (n=4). Panel (E) shows Real-time PCR demonstrating the expression of primary miR-214 (pri-miR214) and mature miR-214 in LL29 cells treated with DFO (n=3). Data are presented as the means.+-.SEM. ANOVA, followed by uncorrected Fisher's LSD test for A, ANOVA, followed by Tukey's HSD test for B, C and student's t-test for E.
[0018] FIG. 10. FENDRR overexpression attenuates bleomycin-induced mouse lung fibrosis. On day 0, mice were infected with AdFENDRR or AdCON (5.times.10.sup.9 IU) through nasal instillation. On day 1, saline (Sal) or bleomycin (Bleo) (1 U/kg BW) was delivered into the mouse lungs through nasal instillation. At D14, the mice were subjected to an analysis of respiratory mechanics by Flexivent and then sacrificed. The left lungs were collected for RNA and protein analysis, and the right lungs were fixed for histological analysis. Panel (A) shows Real-time PCR demonstrating FENDRR overexpression in mouse lungs. Primers detecting human FENDRR transcript variants 2 and 3 and murine Fendrr transcript variants 1 and 2 were used. Sal+AdCON (n=5), Sal+AdFENDRR (n=7), Bleo+AdCON (n=6), Bleo+AdFENDRR (n=8). Panel (B) shows H&E staining demonstrating fibrotic changes in mouse lungs induced by bleomycin. FENDRR attenuated the fibrotic changes in bleomycin-treated mouse lungs. Scale bar: 100 .mu.m. Panel (C) shows Ashcroft score demonstrating that FENDRR lung transfer attenuated bleomycin-induced mouse pulmonary fibrosis. Sal+AdCON (n=15), Sal+AdFENDRR (n=20), Bleo+AdCON (n=21), Bleo+AdFENDRR (n=24). Panel (D) shows a Hydroxyproline assay. Sal+AdCON (n=5), Sal+AdFENDRR (n=7), Bleo+AdCON (n=6), Bleo+AdFENDRR (n=7). Panels (E, F) show Real-time PCR analysis demonstrating that FENDRR inhibited bleomycin-induced Col1a1 and Col3a1 mRNA expression levels in mouse lungs. Sal+AdCON (n=7), Sal+AdFENDRR (n=12), Bleo+AdCON (n=14), Bleo+AdFENDRR (n=16). Panels (G, H) show Western blotting demonstrating that FENDRR inhibited bleomycin-induced COL1A1 protein expression in mouse lungs. Sal+AdCON (n=6), Sal+AdFENDRR (n=6), Bleo+AdCON (n=6), Bleo+AdFENDRR (n=6). Panel (I) shows that FENDRR improved respiratory function in bleomycin-treated mouse by Flexi-vent analysis. Elastance (Ers) was measured in a single-compartment model. Sal+AdCON (n=6), Sal+AdFENDRR (n=7), Bleo+AdCON (n=6), Bleo+AdFENDRR (n=7). The results are presented as the means.+-.SEM. ANOVA, followed by Tukey's HSD test for A, C, E, F, H and ANOVA, followed by uncorrected Fisher's LSD test for D, I.
[0019] FIG. 11. Schematic representation of one, non-limiting mechanistic model for the regulation of FENDRR in idiopathic pulmonary fibrosis.
DETAILED DESCRIPTION
[0020] Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary language and results, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. The inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary--not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
[0021] Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.
[0022] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this presently disclosed inventive concept(s) pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.
[0023] All of the compositions and/or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the inventive concept(s) have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the inventive concept(s). All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the inventive concept(s) as defined by the appended claims.
[0024] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
[0025] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."
[0026] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.
[0027] The use of the term "or" in the claims is used to mean an inclusive "and/or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
[0028] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.
[0029] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition/apparatus/device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.
[0030] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
[0031] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
[0032] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. For example, the term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.
[0033] The term "polypeptide" as used herein will be understood to refer to a polymer of amino acids. The polymer may include d-, I-, or artificial variants of amino acids. In addition, the term "polypeptide" will be understood to include peptides, proteins, and glycoproteins.
[0034] The term "polynucleotide" as used herein will be understood to refer to a polymer of two or more nucleotides. Nucleotides, as used herein, will be understood to include deoxyribose nucleotides and/or ribose nucleotides, as well as artificial variants thereof. The term polynucleotide also includes single-stranded and double-stranded molecules.
[0035] The terms "analog" or "variant" as used herein will be understood to refer to a variation of the normal or standard form or the wild-type form of molecules. For polypeptides or polynucleotides, an analog may be a variant (polymorphism), a mutant, and/or a naturally or artificially chemically modified version of the wild-type polynucleotide (including combinations of the above). Such analogs may have higher, full, intermediate, or lower activity than the normal form of the molecule, or no activity at all. Alternatively and/or in addition thereto, for a chemical, an analog may be any structure that has the desired functionalities (including alterations or substitutions in the core moiety), even if comprised of different atoms or isomeric arrangements.
[0036] As used herein, the phrases "associated with" and "coupled to" include both direct association/binding of two moieties to one another as well as indirect association/binding of two moieties to one another. Non-limiting examples of associations/couplings include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example.
[0037] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
[0038] The term "pharmaceutically acceptable" refers to compounds, compositions, and/or dosage forms which are, with the scope of sound medical judgment, suitable for administration to humans and/or animals without undue adverse side effects such as (but not limited to) toxicity, irritation, and/or allergic response commensurate with a reasonable benefit/risk ratio.
[0039] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition, excipient, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. The term "pharmaceutically-acceptable carrier" refers to any carrier, vehicle, excipient, and/or diluent known in the art or otherwise contemplated herein that may improve solubility, deliverability, dispersion, stability, and/or conformational integrity of the compositions disclosed herein.
[0040] The term "patient" as used herein includes human and veterinary subjects. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including (but not limited to) humans, domestic and farm animals, nonhuman primates, and any other animal that has mammary tissue.
[0041] The term "child" is meant to refer to a human individual who would be recognized by one of skill in the art as an infant, toddler, etc., or an individual less than about 18 years of age, usually less than about 16 years of age, usually less than about 14 years of age, or even less (e.g., from newborn to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 years of age). The term "elderly" generally refers to a human individual whose age is greater than about 50 years of age, usually greater than about 55 years of age, frequently greater than about 60 years of age or more (e.g., about 65 years of age and upwards).
[0042] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include, but are not limited to, individuals already having a particular condition/disease/infection as well as individuals who are at risk of acquiring a particular condition/disease/infection (e.g., those needing prophylactic/preventative measures). The term "treating" refers to administering an agent to a patient for therapeutic and/or prophylactic/preventative purposes.
[0043] A "therapeutic composition" or "pharmaceutical composition" refers to an agent that may be administered in vivo to bring about a therapeutic and/or prophylactic/preventative effect.
[0044] Administering a therapeutically effective amount or prophylactically effective amount is intended to provide a therapeutic benefit in the treatment, prevention, and/or management of a disease, condition, and/or infection. The specific amount that is therapeutically effective can be readily determined by the ordinary medical practitioner, and can vary depending on factors known in the art, such as (but not limited to) the type of condition/disease/infection, the patient's history and age, the stage of the condition/disease/infection, and the co-administration of other agents.
[0045] The term "effective amount" refers to an amount of a biologically active molecule or conjugate or derivative thereof sufficient to exhibit a detectable therapeutic effect without undue adverse side effects (such as (but not limited to) toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of the inventive concept(s). The therapeutic effect may include, for example but not by way of limitation, preventing, inhibiting, or reducing the occurrence of pulmonary fibrosis. The effective amount for a subject will depend upon the type of subject, the subject's size and health, the nature and severity of the condition/disease/infection to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.
[0046] As used herein, the term "concurrent therapy" is used interchangeably with the terms "combination therapy" and "adjunct therapy," and will be understood to mean that the patient in need of treatment is treated or given another drug for the condition/disease/infection in conjunction with the pharmaceutical compositions of the present disclosure. This concurrent therapy can be sequential therapy, where the patient is treated first with one pharmaceutical composition and then the other pharmaceutical composition, or the two pharmaceutical compositions are given simultaneously.
[0047] The terms "administration" and "administering," as used herein, will be understood to include all routes of administration known in the art, including but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, intratracheal, intrabronchial, mucosal, intramuscular, intraperitoneal, intravitreal, and intravenous routes, and including both local and systemic applications. In addition, the compositions of the present disclosure (and/or the methods of administration of same) may be designed to provide delayed, controlled, or sustained release using formulation techniques which are well known in the art.
[0048] Turning now to the inventive concept(s), certain non-limiting embodiments thereof are directed to an anti-fibrotic composition that comprises an isolated and/or purified Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof and a pharmaceutically-acceptable carrier. In certain non-limiting embodiments, the FENDRR IncRNA or fragment/variant thereof is encoded by a sequence comprising at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids. SEQ ID NO:1 represents Human FENDRR transcript variant 1 (GenBank Accession No. NR_036444), SEQ ID NO:2 is Human FENDRR transcript variant 2 (GenBank Accession No. NR_033925), and SEQ ID NO:3 represents a novel Human FENDRR transcript variant 3 disclosed herein (GenBank Accession No. MK522493.1). SEQ ID NO:133 represents mouse FENDRR transcript variant 1 (GenBank Accession No. NR_130109), while SEQ ID NO:134 represents mouse FENDRR transcript variant 2 (GenBank Accession No. NR045471).
[0049] The fragment/variant of FENDRR IncRNA may be at least about 25 contiguous nucleotides long, such as (but not limited to) at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 425, at least about 450, at least about 475, at least about 500, at least about 525, at least about 550, at least about 575, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 2900, at least about 3000, at least about 3100, or at least about 3200 nucleotides long, or the like. The scope of the present disclosure also includes fragments/variants of FENDRR IncRNA that have a length within a range of two of the above values (i.e., a range of from about 50 to about 500 nucleotides long, a range of from about 100 to about 150 nucleotides long, etc.), as well as a length that falls between two of any of the above values (i.e., at least about 725, at least about 1450, etc.).
[0050] In a particular (but non-limiting) embodiment, the fragment or variant of FENDRR IncRNA may comprise any of the regions outlined in Table 4, such as, but not limited to the region between 1419-1549. However, it will be understood that these "regions" are for purposes of example only, and the fragment or variant may include additional sequence on one or both sides thereof.
[0051] The fragment or variant of FENDRR IncRNA may be encoded by a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids, such as, but not limited to, less than about 29 amino acids, less than about 28 amino acids, less than about 27 amino acids, less than about 26 amino acids, less than about 25 amino acids, less than about 24 amino acids, less than about 23 amino acids, less than about 22 amino acids, less than about 21 amino acids, less than about 20 amino acids, less than about 19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about 16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about 13 amino acids, less than about 12 amino acids, less than about 11 amino acids, less than about 10 amino acids, less than about 9 amino acids, less than about 8 amino acids, less than about 7 amino acids, less than about 6 amino acids, less than about 5 amino acids, and the like.
[0052] The FENDRR IncRNA or fragment or variant thereof may be modified and/or encapsulated so as to improve the stability thereof. Any nucleotide modifications or encapsulation methods known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. For example (but not by way of limitation), the FENDRR IncRNA or fragment or variant thereof may be modified by one of more modifications selected from a PS backbone modification, 2'-OMe, 2'-F, 2'MOE, a sugar substitution, LNA, and/or L-RNA modification.
[0053] In addition (but not by way of limitation), the FENDRR IncRNA or fragment/variant thereof may be encapsulated in a delivery vehicle. Non-limiting examples of delivery vehicles that may be utilized in accordance with the present disclosure include a liposome, a lipoplex, a microvesicle, an exosome, a lipidoid nanoparticle, a polymeric nanoparticle, an inorganic nanoparticle, and a stable nucleic acid particle (SNALP), and the like, as well as variations, derivatives, and combinations thereof.
[0054] Certain non-limiting embodiments of the present disclosure are directed to a pharmaceutical composition that comprises a vector comprising a sequence encoding at least about 100 contiguous nucleotides of a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof and a pharmaceutically-acceptable carrier.
[0055] Any sequences encoding FENDRR IncRNAs or fragments/variants thereof that are known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. In certain non-limiting embodiments, the sequence comprises at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
[0056] Any vectors known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure, so long as the vector allows for expression of the FENDRR IncRNA or fragment/variant thereof. Non-limiting examples of vectors that can be utilized in accordance with the present disclosure include an adenoviral vector, an adeno-associated viral (AAV) vector, an alpha viral vector, a herpes viral vector, a lentiviral vector, a measles viral vector, a pox viral vector, a phage vector, a retroviral vector, and the like.
[0057] The vector can include any other components/elements that aid the vector in functioning in accordance with the present disclosure. For example (but not by way of limitation), the vector may further include an expression control sequence to which the FENDRR sequence is operably linked.
[0058] Any pharmaceutically-acceptable carriers known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. Non-limiting examples include a pharmaceutically acceptable solvent, suspending agent, or vehicle that aid in delivery of the compositions of the present disclosure to the human or animal. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Examples of pharmaceutically acceptable carriers that may be utilized in accordance with the present disclosure include, but are not limited to, PEG, liposomes, ethanol, DMSO, aqueous buffers, oils, DPPC, lipids, other biologically-active molecules, vaccine-adjuvants, and combinations thereof. In addition, in certain particular (but non-limiting) examples, pharmaceutically-acceptable carriers can also contain a physiologically acceptable compound that acts to stabilize the compound and/or increase or decrease the absorption or clearance rates of the pharmaceutical compositions. Physiologically acceptable compounds can include (for example, but not by way of limitation) carbohydrates, such as glucose, sucrose, or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposomal carriers; and/or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds include (for example, but not by way of limitation) wetting agents, emulsifying agents, dispersing agents, and/or preservatives.
[0059] The compositions of the present disclosure may be provided in any form and any formulation that allows the compositions to function in accordance with the present disclosure. For example (but not by way of limitation), the compositions may be in solid (such as, but not limited to, tablets, powders, and dry powder inhalers), liquid (such as, but not limited to, emulsions, microemulsions, solutions, suspensions, syrups, and elixirs), or gels, or sprays, mists, or aerosols.
[0060] Certain non-limiting embodiments of the present disclosure are directed to a method of inhibiting activation of lung fibroblasts. In the method, the lung fibroblasts are contacted with any of the compositions disclosed or otherwise contemplated herein. For example (but not by way of limitation), the composition may be selected from: (i) an isolated and/or purified Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof (as described in detail herein above or otherwise contemplated herein); and/or (ii) a vector comprising a sequence encoding at least about 100 contiguous nucleotides of a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof (as described in detail herein above or otherwise contemplated herein). In particular (but not by way of limitation): in (i), the FENDRR IncRNA is encoded by a sequence comprising at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids; and in (ii), the sequence comprises at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
[0061] Certain non-limiting embodiments of the present disclosure are directed to a method of treating or reducing the occurrence of pulmonary fibrosis (such as, but not limited to, idiopathic pulmonary fibrosis) in a subject. In the method, any of the compositions disclosed or otherwise contemplated herein may be administered to the subject. For example (but not by way of limitation), the composition may be selected from: (i) a composition comprising a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof and a pharmaceutically-acceptable carrier (as described in detail herein above or otherwise contemplated herein); or (ii) a composition comprising a vector and a pharmaceutically-acceptable carrier, wherein the vector comprises a sequence encoding at least about 100 contiguous nucleotides of a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or a fragment or variant thereof (as described in detail herein above or otherwise contemplated herein). In particular (but not by way of limitation): in (i), the FENDRR IncRNA is encoded by a sequence comprising at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids; and in (ii), the sequence comprises at least about 100 contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
[0062] In the methods of the present disclosure, the compositions may be administered in therapeutically effective amounts. An effective amount is a dosage of the composition sufficient to provide a therapeutically or medically desirable result or effect in the subject to which the composition is administered. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the specific route of administration, and like factors within the knowledge and expertise of the health practitioner. For example, in connection with methods directed towards treating subjects having a condition characterized by pulmonary fibrosis, an effective amount would be an amount sufficient to mitigate, reduce, modulate, inhibit, or otherwise effectively treat the condition in the subject.
[0063] Generally, a therapeutically effective amount will vary with the subject's age, condition, and sex, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount is typically, but not limited to, an amount in a range from 0.1 .mu.g/kg to about 2000 mg/kg, or from 1.0 .mu.g/kg to about 1000 mg/kg, or from about 0.1 mg/kg to about 500 mg/kg, or from about 1.0 mg/kg to about 100 mg/kg, in one or more dose administrations daily, for one or more days. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six, or more sub-doses, for example, administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the inhibitors are administered for more than 7 days, more than 10 days, more than 14 days, or more than 20 days. In still other embodiments, the inhibitor is administered over a period of weeks or months. In still other embodiments, the inhibitor is delivered on alternate days. For example, the agent may be delivered every two days, or every three days, or every four days, or every five days, or every six days, or every week, or every month.
[0064] The compounds of the presently disclosed inventive concepts may be administered alone or in combination with one or more additional therapies and may be administered by a variety of administration routes. The particular mode selected will depend, of course, upon the compound selected, the condition being treated, the severity of the condition, whether the treatment is therapeutic or prophylactic, and the dosage required for efficacy. The methods of the present disclosure, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Non-limiting examples of administration routes that may be utilized in accordance with the present disclosure include oral, topical, transdermal, parenteral, subcutaneous, intranasal, intratracheal, intrabronchial, mucosal, intramuscular, intraperitoneal, intravitreal, and/or intravenous routes, and the like.
[0065] For example (but not by way of limitation), the composition can be administered to the pulmonary tract by any methods known in the art or otherwise contemplated herein. For example, but not by way of limitation, commercially available devices are known for many different methods and mechanisms of delivering various liquid or aerosolized pharmaceutical formulations to pulmonary tissue, including (but not limited to), intranasal instillation devices, intratracheal instillation devices, intratracheal injection devices, dry powder inhalers (DPIs), pressurized metered dose inhalers (pMDIs), nebulizers (such as, but not limited to, pneumatic (jet) nebulizers and electromechanical nebulizers), electrohydrodynamic aerosol devices, insufflators, respirators, and the like. These devices can include a single dose or multiple doses of the compositions of the present disclosure.
[0066] In addition, the formulations of the compositions of the present disclosure may include one or more additional components/elements that aid in the administration of the compositions, as based upon the delivery device. For example (but not by way of limitation), pMDIs, DPIs, and nebulizers typically employ one or more propellants to propel the liquid or cloud of dry powder formulation out of the device, to form an aerosol, and/or to atomize the liquid formulation. Any suitable propellants/pressurized gas supplies may be utilized. The propellant may take a variety of forms. For example, the propellant may be a compressed gas or a liquefied gas. Aerosol formulations for use in the subject method typically include (for example, but not by way of limitation) propellants, surfactants, and/or co-solvents. Suitable liquid compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable inhalant solvent such as (but not limited to) isotonic saline or bacteriostatic water. Suitable liquid formulations for nasal sprays or nasal drops typically include (for example, but not by way of limitation) aqueous or oily solutions of the active ingredient.
[0067] When the compositions are formulated for being inhaled, the inhaled formulation may be designed for application to the upper (such as, but not limited to, the nasal cavity, pharynx, and larynx) and/or lower respiratory tract (such as, but not limited to, the trachea, bronchi, and lungs). Different devices and excipients can be used depending on whether the application is to the upper and/or lower respiratory tract and can be determined by those skilled in the art.
Example
[0068] An Example is provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein. Rather, the Example is simply provided as one of various embodiments and is meant to be exemplary, not exhaustive.
[0069] In the present Example, FENDRR was identified as a down-regulated IncRNA in the lungs of IPF patients. In addition, the functional roles and underlying mechanisms of FENDRR in pulmonary fibrosis were further determined. The results of this Example showed that FENDRR inhibited the activation of lung fibroblasts by binding iron-responsive element-binding protein 1 (IRP1) to control iron levels and by competing with the pro-fibrotic miR-214. This Example also demonstrated that FENDRR functions as an anti-fibrotic IncRNA in vivo.
[0070] Materials and Methods
[0071] RNA-seq analyses: Two next-generation RNA sequencing datasets from the lung tissues of IPF patients are publicly available from NCBI's Sequence Read Archive (SRA accession number SRA048904) and NCBI's Gene Expression Omnibus (GEO Series accession number GSE52463). These datasets were re-analyzed to identify altered IncRNAs in IPF lungs. RNAs from 3 normal and 3 IPF patient lungs were sequenced in the SRA048904 datasets. RNAs from 7 normal and 8 IPF patient lungs were sequenced in the GSE52463 datasets. These datasets were re-analyzed to identify altered IncRNAs in IPF lungs. Paired-end reads were directionally mapped to the genomic loci of IncRNA (GRCh37/hg19) by TopHat2 (https://ccb.jhu.edu/software/tophat/manual.shtml). CuffDiff analysis was then performed to identify the dysregulated IncRNAs (cole-trapnell-lab.github.io/cufflinks/). IncRNAs with a log 2-fold change .gtoreq.1 and a False Discovery Rate (FDR)<0.05 were considered to be differentially expressed.
[0072] IPF lung tissues and RNA isolation: Twenty-seven IPF patient lung tissue samples were obtained from Lung Tissue Research Consortium (LTRC). All of the samples were submerged in RNAlater solution and stored at -80.degree. C. before use. Total RNA was isolated from these human lung tissues, murine lung tissues or cells using Tri Reagents (Molecular Research Center, Cincinnati, Ohio). Cytoplasmic and nuclear RNAs were isolated from the cytoplasmic and nuclear fractions of human lung fibroblasts, which were separated using a Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp, Thorold, ON, Canada) (Catalog #21000). The RNA concentration and quality were determined with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Tech., Rockland, Del.) with an A.sub.260/A.sub.280 ratio >1.8 and an A.sub.260/A.sub.230 ratio >1.7.
[0073] Cell culture: HEK 293T cells and human lung fibroblasts (HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, Va., USA). HEK 293A cells were purchased from Invitrogen. HFL1 cells were human diploid fibroblasts derived from fetal lungs. CCD-8Lu and LL29 cells were human fibroblasts isolated from the lungs of a healthy adult and an IPF patient, respectively. The cells were grown and maintained with the following media supplemented with 10% fetal bovine serum (FBS): HFL1, and LL29 cells, F12K Medium (Kaighn's Modification of Ham's F-12 Medium); and CCD-8Lu, Eagle's Minimum Essential Medium. Primary human pulmonary fibroblasts (HPF) were purchased from PromoCell (Heidelberg, Germany). HPF cells were cultured in fibroblast medium with its supplements (PromoCell, Heidelberg, Germany).
[0074] Isolation of mouse lung fibroblasts: Primary lung fibroblasts were isolated from the lungs of saline- or bleomycin-treated mice according to a previously described protocol (1). The cells were cultured in DMEM containing 10% FBS and used at passages 3-9. Alveolar epithelial type II cells were also isolated from these mice and differentiated into alveolar epithelial type I cells, as previously described (2).
[0075] Real-Time PCR
[0076] Relative real-time PCR: The mRNA and IncRNA expression levels were determined by SYBR Green I-based real-time PCR. One microgram of DNase-treated total RNA was reverse-transcribed into cDNA with random primers and oligo dT. The real-time PCR thermal conditions were 95.degree. C. for 10 min, followed by 40 cycles of 95.degree. C. for 15 sec and 60.degree. C. for 60 sec. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or .beta.-actin were used as internal controls. The microRNA expression levels were determined by Real-time PCR as previously described (3). The sequences of the primers used for real-time PCR are shown in Table 1.
TABLE-US-00001 TABLE 1 Primers for Real-Time PCR SEQ ID Name Sequence NO: Human NR_033925-FENDDR- CGCACAGACCCAGGATTACTTC 4 FW (variant 1) NR_033925-FENDDR- GCTCCTTATGCAAGCATTCTTC 5 RE (variant 1) A NR_036444-FENDDR- CCCTGCTCCTCTCGAATTTCT 6 FW (variant 2) NR_036444-FENDDR- CCATGCACCAAATCCTTAAAAT 7 RE (variant 2) GT MK522493-FENDDR- CAGAAGCCCCCTCCTGTTATC 8 FW (variant 3) MK522493-FENDDR- AAGAAGCCAAGCCCATTCTGT 9 RE (variant 3) FENDRR-FW (variant GCGCACAGACCCAGGATTT 10 2, 3) FENDRR-RE (variant ACACGGGCAGAGCTGGTTT 11 2, 3) hCOL1A1-FW CGAAGACATCCCACCAATCAC 12 hCOL1A1-RE CAGATCACGTCATCGCACAAC 13 hCOL3A1-FW TGGCTACTTCTCGCTCTGCTT 14 hCOL3A1-RE TTCCAGACATCTCTATCCGCAT 15 AG h.alpha.SMA-FW GTGTTGCCCCTGAAGAGCAT 16 h.alpha.SMA-RE CGCCTGGATAGCCACATACAT 17 hGAPDH-FW GAAGGTGAAGGTCGGAGTCAAC 18 hGAPDH-RE CATGGGTGGAATCATATTGGAA 19 hACTB-FW GGCACCACACCTTCTACAATGA 20 hACTB-RE ACAGCCTGGATAGCAACGTACA 21 hU2snRNA-FW CATCGCTTCTCGGCCTTTTG 22 hU2snRNA-RE TGGAGGTACTGCAATACCAGG 23 hTFRC-FW ATCCGGTTACTGGGCAATTTC 24 hTFRC-RE TCTGTGTCCTCGCAAAAACAG 25 mature miR-214 ACAGCAGGCACAGACAGGCA 26 pri-miR-214-FW CCCTTTCCCCTTACTCTCCAA 27 pri-miR-214-RE GGATGTTCTGCACAGCAAGT 28 Mouse NR_130109-Fendrr- GAACTCAGGACCTCTGGAAGA 29 FW (variant 1) NR_130109-Fendrr- GGTCTGCCTTGTCGTTTTCTT 30 RE (variant 1) NR_045471-Fendrr- TGCTGAATGGAGGCATCTACA 31 FW (variant 2) NR_045471-Fendrr- GCTTGAACCGTCTCTCCTTTG 32 RE (variant 2) FENDRR-FW (variant CACGATCCCAGGTGGACTTG 33 1, 2) FENDRR-RE (variant TGCAGGAGTGAAGGGTGTCTCT 34 1, 2) mCOL1A1-FW ACGCATGGCCAAGAAGACAT 35 mCOL1A1-RE TTGTGGCAGATACAGATCAAGC 36 A mCOL3A1-FW CACCCTTCTTCATCCCACTCTT 37 mCOL3A1-RE TGACATGGTTCTGGCTTCCA 38 mGAPDH-FW CTCGTCCCGTAGACAAAATGGT 39 mGAPDH-RE TGATGGCAACAATCTCCACTTT 40
[0077] Absolute real-time PCR: The expression levels of FENDRR transcript variants were also determined by using the SYBR Green I-based real-time PCR. Conventional PCR products were amplified and purified with gel extraction, and copy numbers were calculated to construct standard curves (10.sup.3-10.sup.8 copies). A standard curve was plotted with threshold cycle (CT) versus the logarithmic value of the gene copy number. The RNA copy numbers of unknown samples were generated directly from the standard curves with Sequence Detector software provided by the 7500 Fast Real-Time PCR system. All copy numbers were normalized to GAPDH mRNA.
[0078] Droplet digital PCR: Droplet digital PCR: The expression of FENDRR variants were also determined by QX200 AutoDG droplet digital PCR (ddPCR) system (Bio-Rad, Hercules, Calif.) according to the manufacturer's instructions. The ddPCR reaction mixture consisted of the QX200.TM. ddPCR.TM. EvaGreen Supermix, forward and reverse primer (200 nM), and 5 .mu.l cDNA in a total reaction volume of 20 .mu.l Droplets were generated in 8-well cartridges using the automated droplet generator. After completion of the droplet generation, water-in-oil emulsions were transferred to a 96-well plate, and PCR was performed using a C1000 Touch.TM. thermal cycler. Thermal cycling conditions were 95.degree. C. for 5 min, followed by 40 cycles at 95.degree. C. for 30 secs, 60.degree. C. for 1 min with a final 5 min at 90.degree. C. After PCR amplification, fluorescence intensities of each droplet from the samples were measured using the QX200 droplet reader. Positive droplets containing amplification products were distinguished from negative droplets and counted by applying a fluorescence amplitude threshold in QuantaSoft software Version 1.7.4.0917. QuantaSoft software provides copies of a gene per microliter (copies/.mu.l). The number of copies of a gene per template cDNA was calculated as copies/.mu.l.times.20 (.mu.l). The copy number of FENDNR variants were normalized to that of .beta.-actin. The primers used are listed in Table 1.
[0079] Construction of plasmids: FENDRR and miR-214 expression vectors: FENDRR was amplified by PCR using specific primers (Table 2) from human lung tissue cDNA. Mature miR-214 plus 200 bp flanking sequences at each end was amplified by PCR using specific primers (Table 2) from human genomic DNA. The fragments were inserted into adenoviral and lentiviral vectors at XhoI and EcoRI sites, as previously described (4, 5). The control vector was constructed with a random genomic DNA insert with a length of approximately 500 bp that did not contain any known IncRNAs, microRNAs, or mRNAs.
[0080] shRNA vectors: All of the shRNAs were designed by the BLOCK-iT.TM. RNAi Designer software from Invitrogen (Grand Island, N.Y.). shRNAs were inserted into the lentiviral pSIH-H1 vector (System Biosciences, Mountain View, Calif.), which utilizes the H1 promoter to drive shRNA expression. The shRNA sequences are listed in Table 2. A control vector containing scrambled shRNA was purchased from System Biosciences (Mountain View, Calif.).
[0081] FENDRR promoter vector: The 5'-flanking region of FENDRR (-1653 to +90) was amplified by PCR using specific primers (Table 2) from human genomic DNA. The fragments were inserted into the luciferase reporter vector pGL3-Basic (Catalog #E1751, Promega, Madison, Wis.).
[0082] miR-214 sensor vector: DNA containing four copies of the miR-214 binding site was inserted downstream of the firefly luciferase gene using the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, Wis.) at the Nhe I and Sal I sites. pmirGLO also contains the renilla luciferase gene for normalization. The control vector was constructed with a similar-sized random DNA insert that did not contain any known miRNA binding sites. The sensor sequences are listed in Table 2.
[0083] All of the inserts in the plasmid constructs described above were confirmed by DNA sequencing.
TABLE-US-00002 TABLE 2 Primers for the Construction of Plasmids SEQ ID Human NO: microRNA sensor CON-sensor- TCGAGGGGTTCACCGATCCTCCACTGCAGTTGG 41 FW TTCCGCCAGCAGACGAGAACTATTTCCTTAAGT TGTGAAGATCTCTTCGGTAGGCCAGCTGGGTTT TAACATG CON-sensor- AATTCATGTTAAAACCCAGCTGGCCTACCGAAG 42 RE AGATCTTCACAACTTAAGGAAATAGTTCTCGTC TGCTGGCGGAACCAACTGCAGTGGAGGATCGGT GAACCCC miR-214- TCGAGTCTAACTGCCTGTTCCTGCCTGCTGTTA 43 sensor-FW TTACTGCCTGTGTGAGCCTGCTGTACATACTGC CTGTCCAGGCCTGCTGTACATACTGCCTGTATT GGCCTGCTGTG miR-214- AATTCACAGCAGGCCAATACAGGCAGTATGTAC 44 sensor-RE AGCAGGCCTGGACAGGCAGTATGTACAGCAGGC TCACACAGGCAGTAATAACAGCAGGCAGGAACA GGCAGTTAGAC FENDRR overexpression FENDRR-FW TTTCTCGAGCAGACAGCGCGGGCTGGGAG 45 FENDRR-RE TTTGGTCTCGAATTGTCCATCGAGTTGTCATGC 46 TT miR-214 overexpression miR-214-FW TATCTCGAGTTCTGTTACGCAAATTATCCATG 47 miR-214-RE TCTGAATTCATAGGCACCACTCACTTTACTT 48 FENDRR promoter FENDRR-FW TGTTGCTAGCGGGAGGAGGAGGAGGAGGAGGAG 49 FENDRR-RE TGTTCTCGAGGGCAGGTCTGCGTGCGAGCC 50 shRNA Smad2-shRNA- GATCCGCCTGATCTTCACAGTCATCATTCAAGA 51 FW GATGATGACTGTGAAGATCAGGCTTTTTG Smad2-shRNA- AATTCAAAAAGCCTGATCTTCACAGTCATCATC 52 RE TCTTGAATGATGACTGTGAAGATCAGGCG Smad3-shRNA- GATCCGCAACCTGAAGATCTTCAACATTCAAGA 53 FW GATGTTGAAGATCTTCAGGTTGCTTTTTG Smad3-shRNA- AATTCAAAAAGCAACCTGAAGATCTTCAACATC 54 RE TCTTGAATGTTGAAGATCTTCAGGTTGCG FENDRR- GATCCGATTTGCCAGCAACTGCATCATTCAAGA 55 shRNA-FW GATGATGCAGTTGCTGGCAAATCTTTTTG FENDRR- AATTCAAAAAGATTTGCCAGCAACTGCATCATC 56 shRNA-RE TCTTGAATGATGCAGTTGCTGGCAAATCG IRP1-shRNA- GATCCGCCATTGGATCCTGTACAACCTTCAAGA FW1 GAGGTTGTACAGGATCCAATGGCTTTTTG 57 IRP1-shRNA- AATTCAAAAAGCCATTGGATCCTGTACAACCTC 58 RE1 TCTTGAAGGTTGTACAGGATCCAATGGCG IRP1-shRNA- GATCCGCAAATTTGTCGAGTTCTTCGTTCAAGA 59 FW2 GACGAAGAACTCGACAAATTTGCTTTTTG IRP1-shRNA- AATTCAAAAAGCAAATTTGTCGAGTTCTTCGTC 60 RE2 TCTTGAACGAAGAACTCGACAAATTTGCG IRP1-shRNA- GATCCGCCATTACTAGCTGCACAAACTTCAAGA 61 FW3 GAGTTTGTGCAGCTAGTAATGGCTTTTTG IRP1-shRNA- AATTCAAAAAGCCATTACTAGCTGCACAAACTC 62 RE3 TCTTGAAGTTTGTGCAGCTAGTAATGGCG
[0084] Production of lentiviruses and adenovirus: Lentiviruses were produced using the Lenti-X.TM. HTX Packaging vectors (Clontech, Mountain View, Calif.) in HEK 293T cells. For the production of adenovirus, a pENTR vector was switched into the adenoviral vector pAd/PL-DEST using the gateway technique (Invitrogen). The obtained adenoviral vector was linearized by Pac I and transfected into HEK 293A cells to produce virus. Adenovirus was further amplified by re-infecting HEK 293A cells. The adenoviruses were concentrated and purified with the Adenovirus Standard Purification ViralKit.TM. (VIRAPUR, San Diego). The viral titers were determined by infecting HEK 293T or HEK 293A cells with a series of dilutions of the viral stock and counting the numbers of virus-infected green fluorescent protein (GFP)-positive cells.
[0085] Generation of fibroblasts stably expressing FENDRR: Stable cell lines expressing FENDRR were generated using a lentiviral FENDRR vector coupled with puromycin selection. LL29 cells were treated with lentiviral FENDRR or the virus control at a multiplicity of infection (MOI) of 50. After 48 hours, cells were selected with puromycin at 0.5 .mu.g/ml for 1 week until they reached confluence, and the medium was changed every two days. The stable cell lines were then cultured with a maintenance concentration of puromycin (0.1 .mu.g/ml) and directly used for further experiment. The stable cell lines were used at passages 4-5.
[0086] Dual luciferase assay: For the promoter luciferase reporter assay, HPFs and HFL1 cells were seeded onto a 96-well plate at a density of 2.times.10.sup.4 cells per well and transfected with 50 ng of the FENDRR promoter reporter vector and TK plasmid (15 ng), which expresses Renilla luciferase for normalization, using Lipofectamine.TM. 2000 (Invitrogen). 24 hours post transfection, cells were treated with TG.beta.31 (5 ng/ml). For the miR-214 sensor luciferase assay, 1) HEK293T cells were seeded onto a 96-well plate at a density of 2.times.10.sup.4 cells per well and transfected with 5 ng miR-214 sensor and 20, 40, or 60 ng miR-214 expression or control vector and 150 ng FENDRR expression or control vector using Lipofectamine.TM. 2000. 2) LL29 cells stably expressing FENDRR or control were seeded onto a 96-well plate at a density of 2.times.10.sup.4 cells per well and transfected with 50 ng miR-214 sensor or control sensor using Lipofectamine.TM. 2000. The cells were collected 48 hours after transfection, and firefly and Renilla luciferase activities were measured using the Dual-Luciferase.RTM. Reporter Assay System (Promega, Madison, Wis.). The results are presented as the ratio of firefly to Renilla luciferase activities.
[0087] RNA pulldown and mass spectrometry analysis: FENDRR with the SP6 promoter were PCR-amplified from FENDRR overexpression vector to serve as DNA template. Antisense FENDRR was amplified as a control (Table 3). The DNA templates were in vitro transcribed to obtain FENDRR and control RNA by using an in vitro transcription kit (ThermoFisher Scientific Catalog #AM1330). The RNAs were labeled using a Pierce RNA 3' End Desthiobiotinylation Kit, and the proteins that interacted with the FENDRR RNA were immunoprecipitated using a Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific Catalog #20163 and 20164). The RNA pull-down protein samples were analyzed by mass spectrometry (LTQ Orbitrap XL).
TABLE-US-00003 TABLE 3 Primer Sequences for RNA Pulldown SEQ ID Name Sequence NO: SP6-FENDRR-FW ATTTAGGTGACACTATAGAAGAGCA 63 (variant 3) GACAGCGCGGGCTG SP6-FENDRR-RE ATATCTATATATGCAAATTAGATGT 64 (variant 3) CTAAATCTATATTCG SP6-anti-sense ATTTAGGTGACACTATAGAAGAGCA 65 FENDRR-FW AATTAGATGTCTAAATCTATATTC (variant 3) SP6-anti-sense CAGACAGCGCGGGCTGGGAG 66 FENDRR-RE (variant 3)
[0088] RNA immunoprecipitation (RIP) assay: RIP was carried out as described previously (6). Confluent LL29 cells in 10-cm dishes were harvested by trypsin digestion and were lysed in RIP buffer. The cell lysate was centrifuged at 10,000.times.g for 15 minutes, and the supernatant was collected. Ten .mu.g of rabbit anti-aconitase 1 (ACO1) (also named IRP1) antibodies (catalog no. Ab126595, Abcam, Boston, Mass.) or IgG control antibodies were added to 50 .mu.l supernatant, and the mixture was incubated overnight with rotation at 4.degree. C. Forty microliters of protein A/G beads were added and incubated at 4.degree. C. for 1 hour with gentle rotation. The beads were washed three times with ice-cold RIP buffer and three times with PBS. The co-precipitated RNAs were isolated from the re-suspended beads in 1 mL Tri Reagents (Molecular Research Center). The amount of FENDRR in the co-precipitated RNAs was determined by real-time PCR and was calculated with the equation 2.sup.-Ct. Enrichment fold was calculated over IgG control.
[0089] Cross-linking immunoprecipitation and qPCR (CLIP-qPCR) analysis: CLIP-qPCR was performed as previously described (7). LL29 cells were cultured in 10-cm dishes until confluence. Cross-linking was performed by irradiating the cells on dishes with 150 mJ/cm.sup.2 of UVA in Spectrolinker. The cell pellets were resuspended in three volumes (relative to pellet size) of NP-40 lysis buffer (7). Cell lysates were digested with 1 U/.mu.l of RNase T1 (ThermoFisher Scientific Catalog #AM2280) for 2, 4, 8, and 12 min. The samples treated with RNAse T1 for 2 min having RNAs partially digested in the 100- to 300-nt range in 1.5% formaldehyde agarose gel were selected. One ml of cell lysates was added to the Sepharose beads coated with 10 .mu.g of normal IgG or anti-IRP1 (ACO1) antibodies and incubated for 3 hours at 4.degree. C. The beads were washed three times with NP-40 lysis buffer and incubated with 20 units of RNase-free DNase I in 100 .mu.l NP-40 lysis buffer for 15 min at 37.degree. C. The beads were then incubated with 0.1% SDS and 0.5 mg/ml Proteinase K for 15 min at 55.degree. C. RNA was isolated with acidic phenol extraction and ethanol precipitation. Real-time PCR was used to quantify the relative abundance of overlapping segments spanning the FENDRR RNA. For primer design, FENDRR was divided into 200-nt overlapping intervals so that the amplified products covered all of the full-length transcript. The sequences of the primers used for real-time PCR are shown in Table 4.
TABLE-US-00004 TABLE 4 Primers for CLIP-qPCR SEQ Primer ID Region name Primer sequence NO: 134-257 FW1 CGAAAGGTGGTGCCGAGAG 67 134-257 RE1 TCCGTTTGCATCCAACATTGT 68 195-294 FW2 GCACAGACCCAGGATTTGTG 69 195-294 RE2 CAGAGCTGGTTTTGACAGTGA 70 241-382 FW3 TGTTGGATGCAAACGGATTTG 71 241-382 RE3 CCCTCTCTGGTCTTCAGTTTCT 72 301-413 FW4 TCTTCCGAAGATACCAAGTGAAA 73 301-413 RE4 GTCAGTTGTGCCAAACTGAGT 74 373-511 FW5 CCAGAGAGGGTGAGTGGTTTA 75 373-511 RES TGCAGTTCCTGTAGGTCAGAA 76 474-587 FW6 CCCTGCTCCTCTCGAATTTCT 77 474-587 RE6 TTTCTGGTTATCTACGACTGCAT 78 540-704 FW7 CCACTGCATTTTGGCATGATT 79 540-704 RE7 GCACACTGCTCAGAGAATGTG 80 649-768 FW8 CCACCAATTGGCTCGATGAG 81 649-768 RE8 GTGACCTGTGAGTGGCGATAA 82 732-857 FW9 CAGAAGCCCCCTCCTGTTATC 83 732-857 RE9 AAAGAAGCCAAGCCCATTCTG 84 837-962 FW10 CAGAATGGGCTTGGCTTCTTT 85 837-962 RE10 AGCCTATGTCCCATCAACAGT 86 909-1058 FW11 CGTTTGTTCATTTTCACCACCAT 87 909-1058 RE11 CCTCCAACAGAAATGCATGCA 88 1001-1149 FW12 CCAGCTGGAGACTGGTATATGT 89 1001-1149 RE12 GCACCAAATCCTGAGAAAGAAGA 90 1086-1227 FW13 CAGACCACCCTCAAATTGAGT 91 1086-1227 RE13 CCAGTTGAGCCTCTGAATGAC 92 1204-1345 FW14 GCAGTCATTCAGAGGCTCAAC 93 1204-1345 RE14 CACAGGGCAAGGATACAGAGA 94 1312-1445 FW15 TCCTCAGCTCACCTCTCTGTA 95 1312-1445 RE15 AGTGACAGTCTGGGACATCTG 96 1419-1549 FW16 AGGATTCAGATGTCCCAGACT 97 1419-1549 RE16 AAGATGATCCCCAACAATGCT 98 1496-1642 FW17 CAGGAATGGGTGGCATATGC 99 1496-1642 RE17 TCTGAGCACACAATCCACTCT 100 1596-1738 FW18 AGGAAGTCCACTATGCTTGCT 101 1596-1738 RE18 CCTTGATTCACAATGGCTCAGT 102 1715-1847 FW19 GCACTGAGCCATTGTGAATCA 103 1715-1847 RE19 CTGGTTTGTGGAGGAGAGAGT 104 1812-1933 FW20 TTGGATTCCAGGGGCACTCT 105 1812-1933 RE20 GCCACTCACATTGTTGCTGAA 106 1903-2043 FW21 GATCCTGCTGTTCAGCAACAA 107 1903-2043 RE21 ACTGCAAACCAAAAGGTGCTT 108 1995-2118 FW22 CCGGTCACTTCACGATGACA 109 1995-2118 RE22 AACTTCTCCAAGCACAAAGTGT 110 2071-2246 FW23 CGGCTTCAGGAATTCACAGAA 111 2071-2246 RE23 CCTAGGTTGTCTCCACTGCTA 112 2178-2319 FW24 GGAGGTCCTGTGTATGAGGAT 113 2178-2319 RE24 ACTGCTGAACACACTTTTCCT 114 2272-2437 FW25 GAGCCCTACAGCAGTGAAAAG 115 2272-2437 RE25 ACCTGGGCATTTACCTTCAGA 116 2372-2532 FW26 GAACTAGGGTAGAGGCACTAGAG 117 2372-2532 RE26 CCCCCTTTCCTGACTACTTAACT 118 2481-2649 FW27 TCACCTGACCTCTGTGTTTACA 119 2481-2649 RE27 GCATATTCATGGCAGCTGGTA 120 2580-2739 FW28 AGTCCCCAAAAACAGGTGGAA 121 2580-2739 RE28 ACAGGCATGTTTTCTTTCCTAGA 122 2654-2816 FW29 GCTGCTGGCTAGTGATAAATAAC 123 2654-2816 RE29 CTCTGTGCCCATCTCTACCAT 124 2758-2901 FW30 AGCTTAGACAAAGATGGGCAAA 125 2758-2901 RE30 CATTCCCCGGCTTGTAAAGG 126 2835-2991 FW31 CACAAACAGAAGGCCAAGTGA 127 2835-2991 RE31 AAAGGTGATGAAGGGCCAGTT 128 2949-3085 FW32 CTCCGTCAGAGTCTCCAGAAG 129 2949-3085 RE32 CGTTTTCTGTGGCAACCATAAC 130 3007-3174 FW33 CAGAACTGGGCAAGAAAATGTTT 131 3007-3174 RE33 GTTGTCCATCGAGTTGTCATG 132 *The primers detect human FENDRR variant 3.
[0090] Iron levels and aconitase activity assay: Iron levels in the fibroblasts and lung tissues were determined by using an Iron Assay Kit purchased from Sigma (Catalog #MAK025) and expressed as nmol/mg protein. Aconitase activity was measured by using an Aconitase Enzyme Activity Microplate Assay Kit purchased from Abcam (Cata #ab109712) according to the manufacturer's instructions and expressed as nmol/min/mg protein.
[0091] Gel contraction activity: Gel contraction activities were measured as previously described (8). LL29 cells stably expressing FENDRR or control vector were split into 6-well plates (200,000 cells/well). The cells were treated with TGF.beta.1 (5 ng/ml) for 72 hours. Then, the cells were mixed with collagen I (MilliporeSigma, Burlington, Mass.) to a final density of 1.times.10.sup.5 cells per ml and a final concentration of 1 mg/ml of collagen I with or without 5 ng/ml of TGF.beta.1. Gel images were taken at 24 hours and quantitatively analyzed using Image J software.
[0092] Western blotting: Protein samples were separated on 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Primary antibodies and dilutions used for Western blotting included: rabbit anti-SMAD2 (catalog no. 3122S, 1:1000 dilution, Cell signaling, Beverly, Mass.); rabbit anti-SMAD3 (catalog no. 9523S, 1:1000 dilution, Cell signaling); rabbit anti-COL1A1 (catalog no. sc-8784-R, lot #J3013, 1:500 dilution, Santa Cruz Biotechnology, California, CA); rabbit anti-COL3A1 (catalog no. sc-8780-R, lot #L1010, 1:500 dilution, Santa Cruz Biotechnology); mouse anti-.alpha.-SMA (catalog no. A2547, 1:1000 dilution, Sigma, St Louis, Mo.); and mouse anti-GAPDH (catalog no. ab181602, 1:4000 dilution, Abcam, Boston, Mass.). The secondary antibodies (horseradish peroxidase-conjugated Immuno-Pure anti-rabbit or mouse IgG; HOUR+L) were used at a dilution of 1:5000. The blots were developed with Super Signal West Pico Luminol Enhancer solution and Super Signal West Pico Stable Peroxidase solution (ThermoFisher Scientific).
[0093] Immunocytochemistry: LL29 cells stably expressing FENDRR or control vector were cultured and maintained with 0.1 .mu.g/ml of puromycin. Cells were split into 8-chamber slides at a density of 4,000 cells per chamber for 24 hours. The cells were stimulated with 5 ng/ml of TGF-.beta.1 for 48 hours, and then the cells were fixed for immunocytochemical analysis with an anti-.alpha.-SMA antibody (1:500) (Sigma) and Alexa 546-conjugated second antibody (1:300) (Molecular Probes, Eugene, Oreg.). Nuclei were stained with Hoechst 33342 (2 .mu.g/ml).
[0094] A mouse model of bleomycin-induced pulmonary fibrosis: The animal procedures were approved by the Institutional Animal Care and Use Committee at Oklahoma State University. C57BL/6 male mice (8-10 weeks old) were randomly divided into four groups: saline (Sal) and control adenovirus (AdCON), saline and FENDRR adenovirus (AdFENDRR), bleomycin (Bleo) and control adenovirus, and bleomycin and FENDRR adenovirus. On day 0, 60 .mu.l of FENDRR or control adenovirus (5.times.10.sup.9 infectious units (IU) per mouse) were delivered into the lung intranasally. On day 1, 60 .mu.l of bleomycin (1 U per kg body weight) (Sigma cat #B8416-15UN) or saline (Sal) was delivered intranasally. On day 14, respiratory mechanics were determined using the FlexiVent (Scireq, Montreal, Canada). Then, the left lung was collected for RNA, protein and collagen content analyses. The right lung was fixed in 4% paraformaldehyde for histological analysis. The degree of fibrosis in the mouse lung was quantitated using an Ashcroft score in a blinded manner following the published method (9).
[0095] Hydroxyproline Assay: The amount of collagen in the lung tissues was determined by the hydroxyproline assay according to the manufacturer's protocol (QuickZyme Biosciences, Netherland) and expressed as .mu.g per mg lung tissue.
[0096] Statistical analysis: The data presented in the figures represent the means.+-.standard error (SEM). Statistical analyses were performed using Student's t-test for two-group comparisons, and Analysis of variance (ANOVA), followed by Tukey's HSD test or Fisher's LSD test, for multiple comparisons. A p-value <0.05 was considered to be significant.
[0097] Results
[0098] FENDRR is down-regulated in fibrotic lungs and fibroblasts.
[0099] There are 2 publicly available RNA_seq datasets from IPF patient lungs: SRA048904, 3 IPF cases and 3 controls (34), and GSE52463, 8 IPFs and 7 controls (35). To identify dysregulated IncRNAs in the lungs of IPF patients, the datasets were re-analyzed for IncRNA expression. 174 (80 up and 94 down) and 56 (6 up and 50 down) dysregulated IncRNAs in IPF lungs were identified from the SRA048904 and GSE52463 datasets, respectively. Of these, 7 down-regulated and zero up-regulated IncRNAs were shared between the 2 datasets (FIG. 1, panel A; Table 5). FENDRR was selected for further studies for the following reasons: (i) FENDRR, but not other IncRNAs (except LINC00961) is conserved between humans and mice; (ii) FENDRR had the fifth and third highest expression among all of the IncRNAs in normal human lungs and human pulmonary fibroblasts (HFPs) (data not shown); and (iii) FENDRR is known to be essential for lung development (21, 23).
TABLE-US-00005 TABLE 5 Seven Common Differentially Expressed IncRNAs in IPF Lungs in the 2 Datasets SRA048904 dataset GSE52463 dataset Gene name Control IPF P value Gene name Control IPF P value RP11-253E3.3 28.11 13.37 0.00165 RP11-253E3.3 11.07 4.08 0.0007 RP11-38P22.2 17.53 8.01 0.00080 RP11-38P22.2 30.14 6.44 0.00005 LINC00961 38.11 15.44 0.00025 LINC00961 11.74 3.06 0.0006 AC093110.3 107.91 40.01 0.00005 AC093110.3 121.79 41.46 0.00025 FENDRR 344.53 89.47 0.00845 FENDRR 147.76 29.19 0.00005 RP1-249H1.4 8.86 1.94 0.00005 RP1-249H1.4 5.70 1.55 0.00005 LINC00551 18.55 2.30 0.00005 LINC00551 7.88 1.00 0.0005
[0100] The down-regulation of FENDRR in the lungs was confirmed using real-time PCR in an independent cohort of 27 patients with IPF from the Lung Tissue Research Consortium (LTRC) and 6 human adult normal lung tissues from BioChain (0.017.+-.0.003 in normal vs. 0.0045.+-.0.001 in IPF) (FIG. 1, panel B). Consistent with these findings using human lung tissues, the Fendrr level in mouse lung tissues with bleomycin-induced pulmonary fibrosis was 40.3.+-.2.4% of the control mouse lungs (FIG. 1, panel C). Fendrr down-regulation was also observed in the fibroblasts, but not in the AEC I and AEC II isolated from the bleomycin-treated mice (FIG. 1, panel D).
[0101] Human FENDRR has two annotated splicing variants, transcript variant 1 (NR_036444; SEQ ID NO:1) and transcript variant 2 (NR_033925; SEQ ID NO:2) in the UCSC Genome Browser (http://genome.ucsc.edu). When it was attempted to clone transcript variant 2 using human lung tissue cDNA, it was found that the cloned FENDRR was 531 bp longer than the transcript variant 2, which is 2,693 bp long (FIG. 2). This was named FENDRR transcript variant 3. The cDNA sequence of variant 3 was submitted to GenBank and assigned the accession number, MK522493.1 (SEQ ID NO:3). Using absolute real-time PCR, it was found that the FENDRR transcript variant 3 was the major transcript in LL29 and HPF fibroblasts (FIG. 1, panel E). Using droplet digit PCR, which provides highly sensitive and absolute quantitation of gene expression, it was found that the FENDRR transcript variant 3 was also the major transcript in normal and IPF human lungs and was reduced in IPF lungs compared to normal lungs (FIG. 1, panel F). Furthermore, variants 1 and 2 were also essentially undetectable in normal and IPF lungs even using the highly sensitive droplet digital PCR technique (FIG. 1, panel F).
[0102] There are two mouse Fendrr variants, which are highly homologous (FIG. 2). The variant 1 was expressed much higher than variant 2 in the mouse lungs. Both transcripts were decreased by bleomycin treatment (FIG. 1, panel G). Human FENDRR transcript variant 3 is positionally conserved to mouse Fendrr transcript variant 1 (FIG. 2) and were used for in vivo mouse studies.
[0103] Unless noted, a primer pair common to human transcript variants 2 and 3 (FIG. 2 and Table 1) were used for detecting human FENDRR expression in this Example. This primer pair was designed and used before the human FENDRR transcript 3 was discovered. Because the human transcript variant 2 is essentially undetectable in human lungs and human fibroblasts (FIG. 1, panels E and F), this primer pair mainly detects the human transcript variant 3.
[0104] TGF.beta.1 inhibits FENDRR expression via Smad3 in pulmonary fibroblasts.
[0105] To assess whether TGF.beta. regulates FENDRR expression, HPFs were treated with TGF.beta.1, and FENDRR levels were determined. TGF.beta.1 reduced FENDRR expression by 42% as measured using the common primer pair to the transcript variants 2 and 3 (FIG. 3, panel A). Similar results were observed using the primer pair specific to the variant 3 and droplet digital PCR (FIG. 3, panel B). Promoter reporter luciferase assays indicated that TGF.beta.1 decreased the FENDRR promoter activity in primary HPFs and HFL1 fibroblasts (FIG. 3, panel C). Next, it was determined whether the TGF.beta.1-mediated inhibition of FENDRR expression occurred through Smad transcription factors using RNA interference. The lentiviral shRNA treatment resulted in 79% and 98% decreases in the protein expression levels of Smad2 and Smad3 in LL29 fibroblasts, respectively (FIG. 3, panel D). The silencing of Smad3 but not Smad2 partially reversed the TGF.beta.1-mediated inhibition of FENDRR expression (FIG. 3, panel E). These results indicate that TGF.beta.1-SMAD3 signaling contributes to the down-regulation of FENDRR in fibrotic lungs.
[0106] FENDRR Inhibits Fibroblast Activation
[0107] FENDRR was overexpressed in IPF LL29 fibroblasts using a lentiviral FENDRR (transcript variant 3) vector coupled with puromycin selection to generate stable cell lines and evaluated the effects of FENDRR overexpression on fibroblast activation. A 13-fold increase in FENDRR levels was observed in the FENDRR-overexpressing cells compared to the virus control (VC) cells (FIG. 4, panel A). FENDRR overexpression reduced TGF.beta.1-induced mRNA and protein expression of .alpha.-SMA, COL1A1, and COL3A1 (FIG. 4, panels B and C). Furthermore, FENDRR overexpression reduced TGF-.beta.1-induced contractile activity, as determined by the collagen gel assay and stress fiber formation (FIG. 4, panels D and E). In contrast, knockdown of FENDRR using shRNA increased the mRNA and protein levels of .alpha.-SMA and the collagens in HPF normal lung fibroblasts (FIG. 4, panels F and G). These results indicated that FENDRR inhibits TGF.beta.-induced fibroblast activation.
[0108] FENDRR Interacts with Iron-Responsive Element-Binding Protein 1
[0109] As a first step in exploring the mechanisms of FENDRR activity, the subcellular localization of FENDRR was determined by measuring the FENDRR levels in the cytoplasmic and nuclear fractions of primary HPFs and LL29 fibroblasts extracted using a Cytoplasmic & Nuclear RNA Purification Kit. FENDRR in both cells had a low ratio of nuclear to cytoplasmic levels (approximately 0.2), similar to those of cytoplasmic GAPDH and ACTB (.beta.-actin) but in contrast to that of nuclear RNA U2 (>2.5) (FIG. 5, panel A), indicating that FENDRR is primarily located in the cytoplasm of fibroblasts.
[0110] As a cytoplasmic IncRNA, the inventors hypothesized that FENDRR may perform its functions by interacting with cytoplasmic proteins. To identify such protein partners, RNA pulldown-coupled mass spectrometry analysis was performed. 29 proteins were enriched in the FENDRR pull-down group with a fold change greater than 2 and a FDR value less than 0.05 compared to the control RNA group (Table 6). These proteins included iron-responsive element-binding protein 1 (IRP1) [also named aconitase1 (ACO1)], which controls iron homeostasis by binding to the iron-responsive element (IRE) of mRNAs related to iron transport and storage (36). Since iron overload is associated with fibrosis (37), IRP1 was selected for further study. First, the interaction of IRP1 and FENDRR in lung fibroblasts was validated. RNA immunoprecipitation analysis showed the robust enrichment of FENDRR in an IRP1-interacting RNA fraction compared to that of an IgG control (FIG. 5, panel B). Moreover, CLIP-qPCR analysis was performed to determine the interaction region between FENDRR and IRP1. The results showed that the primary binding region between FENDRR and IRP1 was located at the 1,419-1,549 bp region (FIG. 5, panel C). RNA secondary structures of FENDRR, predicted using IPknot software, are shown in FIG. 6. The region of FENDRR that interacts with IRP1 involves the integration and formation of two independent RNA structures.
TABLE-US-00006 TABLE 6 FENDRR Binding Proteins Spectrum count of Spectrum count of Protein Control FENDRR Fold Name Description Means SE Means SE change ENOA Alpha-enolase 0.00 0.00 0.74 0.74 N/A IRP1 iron-responsive element-binding protein 1 0.00 0.00 9.60 0.79 N/A (Cytoplasmic aconitate hydratase) GRSF1 Isoform 2 of G-rich sequence factor 1 0.00 0.00 2.10 0.16 N/A CNBP Isoform 2 of Cellular nucleic acid-binding protein 0.09 0.09 2.08 0.88 22.51 ACINU Apoptotic chromatin condensation inducer 0.19 0.09 2.70 1.36 14.44 in the nucleus FXR2 Fragile X mental retardation syndrome-related 0.37 0.37 2.78 1.31 7.53 protein 2 ZO1 Isoform Short of Tight junction protein ZO-1 0.19 0.09 1.40 0.55 7.51 HNRH3 Isoform 2 of Heterogeneous nuclear 0.37 0.19 2.73 0.66 7.30 ribonucleoprotein H3 SRSF9 Serine/arginine-rich splicing factor 9 0.93 0.49 6.53 0.41 7.00 ECI2 Isoform 2 of Enoyl-CoA delta isomerase 2, 0.37 0.37 2.46 1.23 6.68 mitochondrial RBM25 RNA-binding protein 25 1.03 0.56 6.65 1.06 6.49 PRP31 U4/U6 small nuclear ribonucleoprotein Prp31 1.84 0.29 7.05 1.51 3.84 CCAR1 Isoform 2 of Cell division cycle and apoptosis 1.59 0.80 5.71 0.96 3.59 regulator protein 1 SRRM2 Serine/arginine repetitive matrix protein 2 2.20 0.59 7.10 1.04 3.22 IBP7 Isoform 2 of Insulin-like growth factor-binding 1.16 0.66 3.69 0.60 3.16 protein 7 TRA2A Transformer-2 protein homolog alpha 3.55 1.83 10.11 0.54 2.85 RU17 U1 small nuclear ribonucleoprotein 70 kDa 5.98 1.11 16.81 0.03 2.81 RBM14 RNA-binding protein 14 4.87 1.75 12.17 2.39 2.50 K6PP 6-phosphofructokinase type C 1.97 1.07 4.83 1.87 2.45 RNPS1 RNA-binding protein with serine-rich domain 1 1.28 0.65 3.07 0.39 2.41 DHE3 Glutamate dehydrogenase 1, mitochondrial 4.66 1.35 11.19 0.90 2.40 FHL2 Four and a half LIM domains protein 2 3.73 2.23 8.78 3.60 2.35 SRSF4 Serine/arginine-rich splicing factor 4 3.71 0.82 8.72 0.77 2.35 HNRH1 Heterogeneous nuclear ribonucleoprotein H 2.35 0.74 5.50 0.93 2.34 PURB Transcriptional activator protein Pur-beta 2.72 0.62 6.20 1.20 2.28 SNR40 U5 small nuclear ribonucleoprotein 40 kDa protein 1.88 0.64 4.22 1.32 2.25 BUB3 Isoform 2 of Mitotic checkpoint protein BUB3 3.43 1.12 7.42 1.81 2.17 GRP75 Stress-70 protein, mitochondrial 2.21 1.12 4.72 1.57 2.13 SRPK2 Isoform 2 of SRSF protein kinase 2 3.16 0.87 6.52 2.35 2.06
[0111] FENDRR Controls Iron Levels by Suppressing IRP1
[0112] IRP1 is a dual functional protein, with iron regulatory ability and aconitase activity (36, 38). When cellular iron levels are low, IRP1 binds an iron-responsive element (IRE) in either the 3'-untranslated region (UTR) or 5'-UTR of an mRNA to regulate transport and storage of iron. However, when cellular iron levels are high, IRP1 functions as the cytoplasmic isoform of aconitase to catalyze the interconversion of citrate into isocitrate through cis-aconitate. FENDRR overexpression reduced the iron level in LL29 fibroblasts, as determined using an Iron Assay Kit (FIG. 7, panel A), and the aconitase activity, as measured using an Aconitase Enzyme Activity Microplate Assay (FIG. 7, panel B). IRP1 controls iron homeostasis by binding the IRE of mRNAs related to iron transport and storage, including transferrin receptor 1 (TFRC). FENDRR overexpression reduced TFRC mRNA levels (FIG. 7, panel C). The iron levels in fibroblasts isolated from bleomycin-treated mice were higher than those isolated from control mice (FIG. 7, panel D).
[0113] To determine whether FENDRR still regulates cellular iron levels when IRP1 is absent, IRP1 was knocked-down using lentiviral shRNAs, and the effects of IRP1 knock-down on cellular iron levels in the FENDRR-overexpressing fibroblasts was examined. IRP1 protein expression was effectively reduced by a pool of three IRP1 shRNAs (FIG. 7, panel E). Then, cellular iron levels in FENDRR-overexpressed and IRP1-knocked down fibroblasts was measured in the iron-free medium, where IRP1 binds with iron metabolism mRNAs, and the iron-supplemented medium, where IRP1 exists in a free form in cytoplasm. In the iron-free medium, FENDRR reduced cellular iron levels by 64%, and the knock-down of IRP1 rescued FENDRR-mediated reduction in cellular iron levels (FIG. 7, panel E). However, in the iron-supplemented medium, FENDRR reduced only 33% of cellular iron levels, and there were no differences in cellular iron levels between control shRNA and IRP1 knockdown conditions. These results indicate that FENDRR likely competes with the binding of IRP1 to iron metabolism genes under a low cellular iron level.
[0114] Iron is Required for Fibroblast Activation
[0115] Iron overload has been shown to be associated with lung, liver, and renal fibrosis (37, 39). Therefore, the effects of iron on lung fibroblast activation were evaluated. Iron depletion by treatment with the iron chelator desferrioxamine (DFO) suppressed the TGF.beta.1-induced mRNA expression of .alpha.-SMA, COL1A1, and COL3A1 in human lung LL29 fibroblasts (FIG. 7, panels F-H). These results indicated that iron is required for TGF.beta.-induced fibroblast activation.
[0116] FENDRR Competes with Pro-Fibrotic miR-214
[0117] One of the mechanisms for IncRNA function involves sponging of microRNAs. To determine whether FENDRR could act as a competing endogenous RNA (ceRNA) or molecular sponge, RNAhybrid was used to predict potential microRNA binding sites; six binding sites for miR-214-3p on FENDRR were found (1028-1055, 1661-1676, 1876-1914, 2132-2147, 2698-2743, and 3010-3037) (FIG. 8). To validate the predication experimentally, a miR-214 sensor consisting of the firefly luciferase gene and 4 copies of miR-214 binding sites was constructed using the pmirGlo reporter vector. The miR-214 sensor activity was inhibited by endogenous miR-214 in the LL29 fibroblasts, and FENDRR overexpression increased the activity of the miR-214 sensor (FIG. 9, panel A). Then, it was determined whether FENDRR directly competes with miR-214. Transfection of HEK293T cells with a FENDRR expression vector increased the miR-214 sensor activity. However, co-transfection with a miR-214 expression vector reduced the sensor activity in both control and FENDRR overexpression groups in a miR-214-dose-dependent manner (FIG. 9, panel B). These results indicate that FENDRR competes with miR-214.
[0118] miR-214 is up-regulated in the fibrotic lung tissues of IPF patients (40) and in other fibrotic tissues (41-43). miR-214 functions as a pro-fibrotic agent in the kidneys, liver, and heart (41-43). miR-214 was overexpressed in LL29 fibroblasts with a lentiviral vector, and the activation of fibroblasts was examined. miR-214 increased TGF.beta.1-induced COL1A1 mRNA and protein expression (FIG. 9, panels C and D).
[0119] Next, the relationship between iron levels and miR-214 expression was determined. Iron depletion reduced primary and mature miR-214 expression in LL29 cells (FIG. 9, panel E), indicating that iron overload increases miR-214 expression.
[0120] FENDRR Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice
[0121] Since FENDRR inhibits lung fibroblast activation in vitro, the effects of FENDRR overexpression in mouse lungs on bleomycin-induced pulmonary fibrosis in vivo was examined. Adenovirus-mediated gene transfer was also used to overexpress human FENDRR transcript variant 3, which is positionally conserved to mouse fendrr major transcript variant 1. 1.7-fold and 3.5-fold increases in FENDRR expression in the lungs of saline control- and bleomycin-treated mice, respectively, were observed (FIG. 10, panel A). Histopathological analysis showed reduced fibrosis in the FENDRR-treated group (FIG. 10, panel B). Quantitation of lung fibrosis in a blinded manner revealed that increased FENDRR expression significantly decreased the Ashcroft score (FIG. 10, panel C). Furthermore, increased FENDRR expression inhibited lung collagen levels, as measured by hydroxyproline assay (FIG. 10, panel D), and reduced bleomycin-induced COL1A1 and COL3A1 mRNA expression (FIG. 10, panels E and F) and protein expression of COL1A1 (FIG. 10, panels G and H). FENDRR also decreased elastance (Ers), indicating an improvement in lung function (FIG. 10, panel I).
DISCUSSION
[0122] Currently, idiopathic pulmonary fibrosis remains a serious human disease. The lack of clarity surrounding the pathogenesis of IPF has resulted in a lack of effective treatments. In the current study, it was discovered that FENDRR was down-regulated in the fibrotic lungs of IPF patients and bleomycin-treated mice. FENDRR expression was regulated by TGF-.beta./Smad3 signaling. Functionally, FENDRR reduced pulmonary fibrosis by inhibiting fibroblast activation through decreasing cellular iron level via interactions with IRP1 and acting as a ceRNA for profibrotic miR-214 (FIG. 11).
[0123] Fendrr is highly expressed in the adult lung compared with other tissues, and it is confined to the mesenchyme in the developing lungs at E14.5 and E18.5 (44). However, at E9.5, Fendrr is restricted to the caudal end of the lateral plate mesoderm (22). How FENDRR is regulated remains unknown. In the present Example, it was found that FENDRR is down-regulated in fibrotic fibroblasts via TGF.beta.1-SMAD3 signaling. Down-regulation of FENDRR has also been observed in gastric cancer (24). TGF.beta.-SMAD2/3 signaling has been shown to contribute to the pathogenesis of pulmonary fibrosis (45-47). Our current studies demonstrated that TGF.beta.1 inhibited FENDRR promoter activity. Furthermore, knockdown of Smad3, but not Smad2, reversed TG.beta.31-mediated reduction of FENDRR expression, supporting that TGF.beta.1-SMAD3 signaling contributes to the FENDRR down-regulation by TGF.beta.1.
[0124] Among non-coding RNAs, microRNAs have been extensively studied in IPF. However, little is known regarding the roles of IncRNAs in IPF. In this Example, FENDRR was found to have anti-fibrotic functions. In in vitro studies, it was revealed that FENDRR inhibited TGF-.beta.-induced fibroblast activation, as demonstrated by the inhibition of collagen synthesis; reduced .alpha.-SMA mRNA and protein expression; and decreased contractile activity and stress fiber formation. It is noted that FENDRR overexpression had little effects on these parameters under the basal conditions (without TGF.beta.). The possible explanation is that FENDRR may regulate the factors involved in the TGF.beta. signal pathway.
[0125] In in vivo studies, this Example further revealed that adenovirus-mediated FENDRR overexpression reduced collagen content and fibrosis in the lungs in response to bleomycin and improved pulmonary function. Pulmonary fibroblasts express low levels of Coxsackie-virus and adenovirus receptor (CAR). It raises a question whether adenovirus can deliver a gene to fibroblasts. It has been reported that adenoviral vector can be used to deliver ET1 or Fas to mouse and human lung fibroblasts in vitro, respectively (48, 49). Thus, it is possible that this may occur in vivo. However, the possibility that FENDRR is also delivered to the respiratory epithelial cells, which in turn affects pulmonary fibroblasts, was not excluded.
[0126] The inventors discovered that FENDRR was preferentially localized in the cytoplasm of adult lung fibroblasts. FENDRR has been previously shown to predominantly localize in the nucleus during murine lung development (22). However, cellular localization of IncRNAs can be changed under different physiological conditions. For example, IncRNA GASS translocates from the cytoplasm into the nucleus with the glucocorticoid receptor in response to dexamethasone treatment (50). Thus, shifting of FENDRR between the nucleus and cytoplasm might occur during development.
[0127] In a previous study, FENDRR was shown to function in development via epigenetic control mechanisms (20). FENDRR increased the PRC2 occupancy by forming dsDNA/RNA triplexes at target regulatory elements (20). Here, a novel mechanism of FENDRR activity in inhibiting fibroblast activation was demonstrated, i.e., regulating cellular iron levels. Iron is a trace element indispensable for nearly all living organisms, as it participates in a variety of biological processes, including electron transport (51, 52), oxygen transport (53), and DNA synthesis (54). However, excessive iron can result in tissue damage due to the formation of free radicals (55, 56). Abnormal iron homeostasis causes a broad spectrum of human diseases. A number of studies has indicated that iron is associated with pulmonary fibrosis. For example, iron deficiency reduces the severity of bleomycin-induced pulmonary fibrosis in hamsters, possibly due to reduced iron-catalyzed oxygen radical formation, and lipid peroxidation (57). An accumulation of iron after silica instillation causes fibrotic lung injury in rats (58). Mobilization of iron from asbestos with a high percentage of iron can enhance collagen production in rat lung fibroblasts (59). Case studies have shown that interstitial lung disease might be linked to exposure to metal dust (60, 61). Two negative results have also been reported in which the treatment of rats or mice with the iron chelator deferoxamine did not inhibit bleomycin-induced lung fibrosis (62, 63). This lack of effects might have been due to the short period of treatment (62) or, in the long-term study (60 days), with the single dose of bleomycin treatment (63), because single dose bleomycin-induced fibrosis normally resolves spontaneously within 4 weeks of treatment.
[0128] This Example proposes that FENDRR inhibits fibroblast activation by reducing iron levels via interacting with IRP1, which was supported by the following observations: (i) IRP1 was identified as the target protein of FENDRR by RNA pulldown-coupled mass spectrometry analysis; (ii) FENDRR overexpression inhibited ACO1 activity, reduced cellular iron levels, and decreased TFRC mRNA expression; and (iii) iron was required for TGF.beta.-induced fibroblast activation.
[0129] How iron regulates lung fibroblast activation is still unclear and needs further studies. A few studies show that iron activates TGF.beta. signaling. One study shows that iron activates TGF.beta. signaling by increasing TGF.beta. RII receptor and the phosphorylation of Smad2 in murine hepatic stellate cells (64). Another study reports that iron chelators inhibit the TGF.beta./Smad pathway in prostate and colon cancer cells via the reduction in Smad2 expression and Smad3 phosphorylation due to an increase in N-myc downstream-regulated gene-1 (NDRG1) expression (65). Thus, it is possible that FENDRR reduces iron levels, which in turn inhibits TGF.beta. signaling.
[0130] This Example also demonstrated another new mechanism for FENDRR activity regarding its anti-fibrotic effects, i.e., suppressing pro-fibrotic miR-214 activity by acting as its ceRNA. A growing body of evidence indicates that IncRNAs can act as ceRNAs to sponge microRNAs (66). A previous publication also indicated that IncRNAs function in pulmonary fibrosis by interacting with microRNAs to control fibroblast proliferation and activation (67). Here, it is proposed that FENDRR inhibits fibroblast activation by sponging miR-214. This is supported by the following observations: (i) miR-214 enhanced fibroblast activation; (ii) FENDRR contains six binding sites for miR-214; (iii) FENDRR overexpression increased the activity of a miR-214 sensor; and (iv) FENDRR directly competed with miR-214 to affect miR-214 sensor activity. However, a rescue experiment is desired to address if miR-214 can reverse the anti-fibrotic activity of FENDRR. Furthermore, the relative contributions of iron-IRP1 and miR-214 to FENDRR activities remain to be determined. However, iron depletion reduces miR-214 expression, indicating that these two pathways may cross-talk.
[0131] In summary, this Example demonstrated that the IncRNA FENDRR is an antifibrotic IncRNA in the lung. This Example further demonstrated that regulating cellular iron levels and competing with miR-214 are two novel mechanisms underlying FENDRR activity in pulmonary fibroblasts.
[0132] Thus, in accordance with the present disclosure, there have been provided compositions, and kits containing same, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.
Sequence CWU
1
1
14112693DNAHomo sapiens 1agcagacagc gcgggctggg agcgccggca agggcggccc
tcgcgcacct ggctgccagc 60ccgcaggggg ctcgcacgca gacctgccct tgccacctca
gcgctcgggt gcgggctggg 120ggcggtggag ccacgaaagg tggtgccgag agcggagcgc
aggacagcgc aggagccccg 180gcgcatcgcg gcgcgcacag acccaggatt tgtgaagagg
tgacccaaat ttcaagacaa 240tgttggatgc aaacggattt gccagcaact gcatcactgt
caaaaccagc tctgcccgtg 300tcttccgaag ataccaagtg aaatacatgt agatgggatt
tacatctgaa aaaccaagtg 360agaaactgaa gaccagagag ggtgagtggt ttactcagtt
tggcacaact gaccccaacc 420tagccctcca tgaggactga gcgcatgaga gatcctgagc
cacagccgcc cagccctgct 480cctctcgaat ttctgaccta caggaactgc aagaagtaat
gaaagactgc tgtttaaagc 540cactgcattt tggcatgatt tgttatgcag tcgtagataa
ccagaaaaca tcggatttac 600attttaagga tttggtgcat gggatgggct cagttgggca
gttcttctgt tccatgtgat 660ggtactgggg ctgcagtcat tcagaggctc aactgggctg
aacgtgcaag gtttcttgct 720cacgtggcca tcagtggtgc tggctgctgt tgggagctcc
actggacttc tgtccagagc 780tcctcagctc acctctctgt atccttgccc tgtggctcag
gcttctaaca atacagaggc 840tggattccaa gaggagggaa ttagaagcgt tcagccttct
taagaccagg attcagatgt 900cccagactgt cactgtattg accggagcat ttacaggcca
gcctagtaat ttttccatct 960tgggcaggaa tgggtggcat atgctggggg taggggtagc
attgttgggg atcatctttg 1020gagctgtgtg ccaaggagta aattgctggg ctgctttcta
cctgaggaag tccactatgc 1080ttgcttgtgg agagtggatt gtgtgctcag agtccacaag
gatttaagaa tccgagccct 1140gctgcccttg cctcagcctg tgcattaggg cccggtggtg
agagcactga gccattgtga 1200atcaaggccc ctccctcgac ctgcatacta acaggccaga
agagcttgct tgattttggt 1260cctaggctcc tgcctgtggg ttggattcca ggggcactct
ctcctccaca aaccagacgg 1320tggcaggtac atcccctcaa tggaaattgc ccacacttca
ccctaaagac agatcctgct 1380gttcagcaac aatgtgagtg gcccacacat ttgtccagca
tgaaaccgtt tgcagattcc 1440tttccagtac acagtcccgt cagccggtca cttcacgatg
acagctcctc gaagcacctt 1500ttggtttgca gttgtgtagc ttagaagtag gagcctgcac
ggcttcagga attcacagaa 1560ggagcacact ttgtgcttgg agaagttaat tataggaata
tcctgcaacc tgctgctctg 1620agcctgtgca cctggggtgt ttccctggag gtcctgtgta
tgaggatact catcagcatg 1680gggtttggga agcctagcag tggagacaac ctaggtggtc
ctctccaggg gagtaggtaa 1740gagccctaca gcagtgaaaa gcattgaagg aaaagtgtgt
tcagcagtgc tcttaatctg 1800atcagagcag cctgaacaca ttctaaacac agggcattta
gaactagggt agaggcacta 1860gagcaccata gatacaaact cagattctga aggtaaatgc
ccaggttcaa atccaagccc 1920cctcgccaag tgaccatgac tcccccgagt cacctgacct
ctgtgtttac atcctcaaag 1980ttaagtagtc aggaaagggg gcttggggct tgccacatgg
atggttgcca ctctcctttg 2040caaagtgtag tccccaaaaa caggtggaag agggaagggt
ccaattagaa ggccgagtac 2100cagctgccat gaatatgcca gagctgctgg ctagtgataa
ataactgaaa cccaggggga 2160cagcagagct tgggtgacca tacattctag gaaagaaaac
atgcctgttg tcctggcttt 2220ttatccagct tagacaaaga tgggcaaaga aaataattta
gaaaatggta gagatgggca 2280cagagaaagg gcacggaggg aggcacaaac agaaggccaa
gtgatggtgg aggcagacat 2340tggagaaatg cctttacaag ccggggaatg ccaaggatgc
tggcaaccct gggagtggga 2400agaacctgga acacaccctc cgtcagagtc tccagaagga
actggccctt catcaccttt 2460cggacttctg gcctccagaa ctgggcaaga aaatgtttct
gctgttggag gccatccagc 2520tggtggtcat ttgttatggt tgccacagaa aacgaatata
gatttagaca tctaatttgc 2580atatatagat atgcatgtgt atttgttaaa taaaagataa
agcatgacaa ctcgatggac 2640aacgttacaa ctcattaaat tgtgttggtt aaaaaaaaaa
aaaaaaaaaa aaa 269323099DNAHomo sapiens 2agcagacagc gcgggctggg
agcgccggca agggcggccc tcgcgcacct ggctgccagc 60ccgcaggggg ctcgcacgca
gacctgccct tgccacctca gcgctcgggt gcgggctggg 120ggcggtggag ccacgaaagg
tggtgccgag agcggagcgc aggacagcgc aggagccccg 180gcgcatcgcg gcgcgcacag
acccaggatt acttcatgca cataaaagaa aacgcaacta 240cagtggaccc tgaacaacct
atggcctgca gccactgaag aatgcttgca taaggagctc 300ttctctgcac ataccccgac
acagtgatgg tatcttgcag ctgctgcttc tgtccaaggc 360actgcagcct actcgtcaaa
agcccgaagc ccagctctgc cagcagtgca ctgtgtgctc 420ttaggaaaca gagaagcaaa
gaagttagca agcttgtttt ggcagaagaa aaaacacaaa 480atacccaacc acagatcctc
aaaaatattg ctaagcacta attttgctaa catgttagac 540agccttcgga tctaggcaat
cttgttcctc agctggaaag agttcctgcc agccatgtga 600ttccaaatcc accctggaca
tatggggata atgagaaaat cagctccggc cttccctggc 660cttccagcca agtgaatggg
ggcatcccct ggtgcttcag cccggggccc acccagagga 720tgctagctgg cctgcagagg
cctcaaccat gccctcctgg actcaggtaa gactgaggtt 780tccatgccct caaaacaatt
gttcagacaa aaactcactg cccaggtcaa agtcacagca 840ccagaaagcc aacctgagga
tgaagacaaa gctctttgtt ggcacagtcc cctgatcatt 900cgaggtcagc atctccatca
aaatggaccc aatgctgagg caagaaggag aacatcactg 960ttaagaaggt aatgagtcac
aaatgctatt tcgatggttt tctttgaaag aaatacacag 1020aaccaaacca attttaatta
aagatcttgt ctttgtaatc aggcagccac aaatgaaggc 1080aaaactgctt tcatgtggca
taatatacac agagctgaac tagttttccc ataaaagggc 1140aggttctttt cttcaaataa
gaaaagagtt tgttcctaac aatatggggg atgcaacaga 1200aagctgtctt ttgcaaagta
taaaattgca gatcctcccg tggaagccat ttcttgatgc 1260taaaccaatg cgaacgtttg
ccaaacgcac atgcgagccg ggattatcct accctttgag 1320attagacatg tgatcctgat
ttttaagtga aaaacgcagc cagaggccaa ataaacatac 1380ctacaagtac atcgaaagct
caaatggttg agaaatgatt gtaacacacc ccggtgtgta 1440tttctgagga agaatcaaca
tgcacatgga aattgatgtt ttgattttta attttaaaac 1500gacgacagaa ctgaaatacc
tcccctaact aggtccactg aaaccagtga ggagataatc 1560attttcagtt aaatatgcat
gtaatttaat aatccacaga tggaaaatgc atttgaggtg 1620gactgaacac agccaggagt
ttttgcttat cgacagcact gaatcctgcc ttgactagag 1680agactcgtca tgtgatcaac
tgtggccaga gggcatcctg cagacagctc ctgtcacacc 1740acactgacca gactcttatg
agtatttaag catcttcagg gaagcgattg actgtcttat 1800aatggtctta caagtacgga
actttattac ctgctaataa tcagagcaca tgatggcaca 1860atagaatata cgagtgaagt
tctaaatgca aggttgttct tattctgtga gtggcaaaac 1920tcatgcagtg agcagataaa
gctggcagga ggcattggcg ccctaatgct gggcactaac 1980cacctgcccg gactgcgaat
atctgttggg tcctttctat atctggtcta ttctcgtgtt 2040tgattatatc ctggtggcaa
gaatagcaaa atcaactctc cttcctatcc catgtggaga 2100ggcatgattt gagtcagaga
gcattatcct ctctgcgtta tccaacccca tgtcccatga 2160tgcactttgt agacaagcaa
gtgtccctgg tggagaatcc acagccacac atcctcattg 2220gttgcaagga tcagtgtcca
atatctactg ccatgatcat tctcagtttt acatccacag 2280agcttcatag aattctaaca
ccacaggtcc aagtagaggt tttggcctca gagtgggcta 2340gattccacct ctgacagcag
ctgtgggact gtttttccat ctgtaaaatg gagacaagag 2400tatcacctac ctcatggtgt
tggtgggatc gttaaacaga attccgatca tgaaaccctt 2460agcctgctcc ctgcataggc
tgcatgcgct cagtgagcta tggctctgtt tcatgctgac 2520tttaccatca tgtaagcagt
tttaaaaatg ctagaagctt tagtcataga attaccatat 2580gatacagcct actgcagagt
ccagaggctg aggcaggaga gttacttgaa cctgggaggc 2640ggaggttgca gtgagccaag
atcgcgccac tgcactccag cctgggcaac agagtgagat 2700tctgtctcaa cacaaaaaag
aagacatgtg tccccatcac agtatcatac agagtgcttc 2760cactgcccta aaataccctg
tgctcttcct attcatccct ctcttcaacc ccagcaacca 2820ctgatccttt taccatctcc
atggttttgc ctttcccaga atgtcatatt gttggaatca 2880tacgctatgt agcctttgca
aatgggcttc tttcaccgag caatgtgcat tgaaggtgcc 2940tctatgactt ttcaagggtt
gatagctcat tcctttttat tgctaaataa tattccattg 3000tctgaatgta ccacattttg
tttatccatt cacctactaa aggacatctt ggttgcttcc 3060aagtttgggc aattataaat
aaaactgcta taaacattg 309933224DNAHomo sapiens
3agcagacagc gcgggctggg agcgccggca agggcggccc tcgcgcacct ggctgccagc
60ccgcaggggg ctcgcacgca gacctgccct tgccacctca gcgctcgggt gcgggctggg
120ggcggtggag ccacgaaagg tggtgccgag agcggagcgc aggacagcgc aggagccccg
180gcgcatcgcg gcgcgcacag acccaggatt tgtgaagagg tgacccaaat ttcaagacaa
240tgttggatgc aaacggattt gccagcaact gcatcactgt caaaaccagc tctgcccgtg
300tcttccgaag ataccaagtg aaatacatgt agatgggatt tacatctgaa aaaccaagtg
360agaaactgaa gaccagagag ggtgagtggt ttactcagtt tggcacaact gaccccaacc
420tagccctcca tgaggactga gcgcatgaga gatcctgagc cacagccgcc cagccctgct
480cctctcgaat ttctgaccta caggaactgc aagaagtaat gaaagactgc tgtttaaagc
540cactgcattt tggcatgatt tgttatgcag tcgtagataa ccagaaaaca tcggatttac
600attttaaggt aaacttttta ttgaaacatc acatacacac agagacgtcc accaattggc
660tcgatgagct ttctcaaagg aaacacattc tctgagcagt gtgcacatcc agacacatca
720tctccatcct ccagaagccc cctcctgtta tcgccactca caggtcaccc gtctcctgac
780ttgcagccac agagatagtc ctgcctaatt ctgaacgtgg tacaaacgga atcatacaga
840atgggcttgg cttctttcac tctgggctgt gtttatgagc atcacccaga ttgctgcatg
900gaagccatcg tttgttcatt ttcaccacca tagagtattc tactgttgat gggacatagg
960ctatttattc cttctactgt tgagggacgc atagttattt ccagctggag actggtatat
1020gtctcttggc taccttctgc atgcatttct gttggaggtt gtttcaggta tctcttaacg
1080tataacagac caccctcaaa ttgagtcctt gaaacaatta ccattttctt ctttctcagg
1140atttggtgca tgggatgggc tcagttgggc agttcttctg ttccatgtga tggtactggg
1200gctgcagtca ttcagaggct caactgggct gaacgtgcaa ggtttcttgc tcacgtggcc
1260atcagtggtg ctggctgctg ttgggagctc cactggactt ctgtccagag ctcctcagct
1320cacctctctg tatccttgcc ctgtggctca ggcttctaac aatacagagg ctggattcca
1380agaggaggga attagaagcg ttcagccttc ttaagaccag gattcagatg tcccagactg
1440tcactgtatt gaccggagca tttacaggcc agcctagtaa tttttccatc ttgggcagga
1500atgggtggca tatgctgggg gtaggggtag cattgttggg gatcatcttt ggagctgtgt
1560gccaaggagt aaattgctgg gctgctttct acctgaggaa gtccactatg cttgcttgtg
1620gagagtggat tgtgtgctca gagtccacaa ggatttaaga atccgagccc tgctgccctt
1680gcctcagcct gtgcattagg gcccggtggt gagagcactg agccattgtg aatcaaggcc
1740cctccctcga cctgcatact aacaggccag aagagcttgc ttgattttgg tcctaggctc
1800ctgcctgtgg gttggattcc aggggcactc tctcctccac aaaccagacg gtggcaggta
1860catcccctca atggaaattg cccacacttc accctaaaga cagatcctgc tgttcagcaa
1920caatgtgagt ggcccacaca tttgtccagc atgaaaccgt ttgcagattc ctttccagta
1980cacagtcccg tcagccggtc acttcacgat gacagctcct cgaagcacct tttggtttgc
2040agttgtgtag cttagaagta ggagcctgca cggcttcagg aattcacaga aggagcacac
2100tttgtgcttg gagaagttaa ttataggaat atcctgcaac ctgctgctct gagcctgtgc
2160acctggggtg tttccctgga ggtcctgtgt atgaggatac tcatcagcat ggggtttggg
2220aagcctagca gtggagacaa cctaggtggt cctctccagg ggagtaggta agagccctac
2280agcagtgaaa agcattgaag gaaaagtgtg ttcagcagtg ctcttaatct gatcagagca
2340gcctgaacac attctaaaca cagggcattt agaactaggg tagaggcact agagcaccat
2400agatacaaac tcagattctg aaggtaaatg cccaggttca aatccaagcc ccctcgccaa
2460gtgaccatga ctcccccgag tcacctgacc tctgtgttta catcctcaaa gttaagtagt
2520caggaaaggg ggcttggggc ttgccacatg gatggttgcc actctccttt gcaaagtgta
2580gtccccaaaa acaggtggaa gagggaaggg tccaattaga aggccgagta ccagctgcca
2640tgaatatgcc agagctgctg gctagtgata aataactgaa acccaggggg acagcagagc
2700ttgggtgacc atacattcta ggaaagaaaa catgcctgtt gtcctggctt tttatccagc
2760ttagacaaag atgggcaaag aaaataattt agaaaatggt agagatgggc acagagaaag
2820ggcacggagg gaggcacaaa cagaaggcca agtgatggtg gaggcagaca ttggagaaat
2880gcctttacaa gccggggaat gccaaggatg ctggcaaccc tgggagtggg aagaacctgg
2940aacacaccct ccgtcagagt ctccagaagg aactggccct tcatcacctt tcggacttct
3000ggcctccaga actgggcaag aaaatgtttc tgctgttgga ggccatccag ctggtggtca
3060tttgttatgg ttgccacaga aaacgaatat agatttagac atctaatttg catatataga
3120tatgcatgtg tatttgttaa ataaaagata aagcatgaca actcgatgga caacgttaca
3180actcattaaa ttgtgttggt taaaaaaaaa aaaaaaaaaa aaaa
3224422DNAHomo sapiens 4cgcacagacc caggattact tc
22523DNAHomo sapiens 5gctccttatg caagcattct tca
23621DNAHomo sapiens 6ccctgctcct
ctcgaatttc t 21724DNAHomo
sapiens 7ccatgcacca aatccttaaa atgt
24821DNAHomo sapiens 8cagaagcccc ctcctgttat c
21921DNAHomo sapiens 9aagaagccaa gcccattctg t
211019DNAHomo sapiens
10gcgcacagac ccaggattt
191119DNAHomo sapiens 11acacgggcag agctggttt
191221DNAHomo sapiens 12cgaagacatc ccaccaatca c
211321DNAHomo sapiens
13cagatcacgt catcgcacaa c
211421DNAHomo sapiens 14tggctacttc tcgctctgct t
211524DNAHomo sapiens 15ttccagacat ctctatccgc atag
241620DNAHomo sapiens
16gtgttgcccc tgaagagcat
201721DNAHomo sapiens 17cgcctggata gccacataca t
211822DNAHomo sapiens 18gaaggtgaag gtcggagtca ac
221922DNAHomo sapiens
19catgggtgga atcatattgg aa
222022DNAHomo sapiens 20ggcaccacac cttctacaat ga
222122DNAHomo sapiens 21acagcctgga tagcaacgta ca
222220DNAHomo sapiens
22catcgcttct cggccttttg
202321DNAHomo sapiens 23tggaggtact gcaataccag g
212421DNAHomo sapiens 24atccggttac tgggcaattt c
212521DNAHomo sapiens
25tctgtgtcct cgcaaaaaca g
212620DNAHomo sapiens 26acagcaggca cagacaggca
202721DNAHomo sapiens 27ccctttcccc ttactctcca a
212820DNAHomo sapiens
28ggatgttctg cacagcaagt
202921DNAMus musculus 29gaactcagga cctctggaag a
213021DNAMus musculus 30ggtctgcctt gtcgttttct t
213121DNAMus musculus
31tgctgaatgg aggcatctac a
213221DNAMus musculus 32gcttgaaccg tctctccttt g
213320DNAMus musculus 33cacgatccca ggtggacttg
203422DNAMus musculus
34tgcaggagtg aagggtgtct ct
223520DNAMus musculus 35acgcatggcc aagaagacat
203623DNAMus musculus 36ttgtggcaga tacagatcaa gca
233722DNAMus musculus
37cacccttctt catcccactc tt
223820DNAMus musculus 38tgacatggtt ctggcttcca
203922DNAMus musculus 39ctcgtcccgt agacaaaatg gt
224022DNAMus musculus
40tgatggcaac aatctccact tt
2241106DNAArtificial sequenceCON-sensor-FW primer 41tcgaggggtt caccgatcct
ccactgcagt tggttccgcc agcagacgag aactatttcc 60ttaagttgtg aagatctctt
cggtaggcca gctgggtttt aacatg 10642106DNAArtificial
SequenceCON-Sensor-RE primer 42aattcatgtt aaaacccagc tggcctaccg
aagagatctt cacaacttaa ggaaatagtt 60ctcgtctgct ggcggaacca actgcagtgg
aggatcggtg aacccc 10643110DNAArtificial
sequencemiR-214-sensor-FW primer 43tcgagtctaa ctgcctgttc ctgcctgctg
ttattactgc ctgtgtgagc ctgctgtaca 60tactgcctgt ccaggcctgc tgtacatact
gcctgtattg gcctgctgtg 11044110DNAArtificial
sequencemiR-214-sensor-RE primer 44aattcacagc aggccaatac aggcagtatg
tacagcaggc ctggacaggc agtatgtaca 60gcaggctcac acaggcagta ataacagcag
gcaggaacag gcagttagac 1104529DNAArtificial
sequenceFENDRR-FW primer 45tttctcgagc agacagcgcg ggctgggag
294635DNAArtificial sequenceFENDRR-RE primer
46tttggtctcg aattgtccat cgagttgtca tgctt
354732DNAArtificial sequencemiR-214-FW primer 47tatctcgagt tctgttacgc
aaattatcca tg 324831DNAArtificial
sequencemiR-214-RE primer 48tctgaattca taggcaccac tcactttact t
314933DNAArtificial sequenceFENDRR-FW primer
49tgttgctagc gggaggagga ggaggaggag gag
335030DNAArtificial sequenceFENDRR-RE primer 50tgttctcgag ggcaggtctg
cgtgcgagcc 305162DNAArtificial
sequenceSmad2-shRNA-FW primer 51gatccgcctg atcttcacag tcatcattca
agagatgatg actgtgaaga tcaggctttt 60tg
625262DNAArtificial
sequenceSmad2-shRNA-RE primer 52aattcaaaaa gcctgatctt cacagtcatc
atctcttgaa tgatgactgt gaagatcagg 60cg
625362DNAArtificial
sequenceSmad3-shRNA-FW primer 53gatccgcaac ctgaagatct tcaacattca
agagatgttg aagatcttca ggttgctttt 60tg
625462DNAArtificial
sequenceSmad3-shRNA-RE primer 54aattcaaaaa gcaacctgaa gatcttcaac
atctcttgaa tgttgaagat cttcaggttg 60cg
625562DNAArtificial
sequenceFENDRR-shRNA-FW primer 55gatccgattt gccagcaact gcatcattca
agagatgatg cagttgctgg caaatctttt 60tg
625662DNAArtificial
sequenceFENDRR-shRNA-RE primer 56aattcaaaaa gatttgccag caactgcatc
atctcttgaa tgatgcagtt gctggcaaat 60cg
625762DNAArtificial
sequenceIRP1-shRNA-FW1 primer 57gatccgccat tggatcctgt acaaccttca
agagaggttg tacaggatcc aatggctttt 60tg
625862DNAArtificial
sequenceIRP1-shRNA-RE1 primer 58aattcaaaaa gccattggat cctgtacaac
ctctcttgaa ggttgtacag gatccaatgg 60cg
625962DNAArtificial
sequenceIRP1-shRNA-FW2 primer 59gatccgcaaa tttgtcgagt tcttcgttca
agagacgaag aactcgacaa atttgctttt 60tg
626062DNAArtificial
sequenceIRP1-shRNA-RE2 primer 60aattcaaaaa gcaaatttgt cgagttcttc
gtctcttgaa cgaagaactc gacaaatttg 60cg
626162DNAArtificial
sequenceIRP1-shRNA-FW3 primer 61gatccgccat tactagctgc acaaacttca
agagagtttg tgcagctagt aatggctttt 60tg
626262DNAArtificial
sequenceIRP1-shRNA-RE3 primer 62aattcaaaaa gccattacta gctgcacaaa
ctctcttgaa gtttgtgcag ctagtaatgg 60cg
626339DNAArtificial
sequenceSP6-FENDRR-FW (variant 3) primer 63atttaggtga cactatagaa
gagcagacag cgcgggctg 396440DNAArtificial
sequenceSP6-FENDRR-RE (variant 3) primer 64atatctatat atgcaaatta
gatgtctaaa tctatattcg 406549DNAArtificial
sequenceSP6-anti-sense FENDRR-FW (variant 3) primer 65atttaggtga
cactatagaa gagcaaatta gatgtctaaa tctatattc
496620DNAArtificial sequenceSP6-anti-sense FENDRR-RE (variant 3) primer
66cagacagcgc gggctgggag
206719DNAHomo sapiens 67cgaaaggtgg tgccgagag
196821DNAHomo sapiens 68tccgtttgca tccaacattg t
216920DNAHomo sapiens
69gcacagaccc aggatttgtg
207021DNAHomo sapiens 70cagagctggt tttgacagtg a
217121DNAHomo sapiens 71tgttggatgc aaacggattt g
217222DNAHomo sapiens
72ccctctctgg tcttcagttt ct
227323DNAHomo sapiens 73tcttccgaag ataccaagtg aaa
237421DNAHomo sapiens 74gtcagttgtg ccaaactgag t
217521DNAHomo sapiens
75ccagagaggg tgagtggttt a
217621DNAHomo sapiens 76tgcagttcct gtaggtcaga a
217721DNAHomo sapiens 77ccctgctcct ctcgaatttc t
217823DNAHomo sapiens
78tttctggtta tctacgactg cat
237921DNAHomo sapiens 79ccactgcatt ttggcatgat t
218021DNAHomo sapiens 80gcacactgct cagagaatgt g
218120DNAHomo sapiens
81ccaccaattg gctcgatgag
208221DNAHomo sapiens 82gtgacctgtg agtggcgata a
218321DNAHomo sapiens 83cagaagcccc ctcctgttat c
218421DNAHomo sapiens
84aaagaagcca agcccattct g
218521DNAHomo sapiens 85cagaatgggc ttggcttctt t
218621DNAHomo sapiens 86agcctatgtc ccatcaacag t
218723DNAHomo sapiens
87cgtttgttca ttttcaccac cat
238821DNAHomo sapiens 88cctccaacag aaatgcatgc a
218922DNAHomo sapiens 89ccagctggag actggtatat gt
229023DNAHomo sapiens
90gcaccaaatc ctgagaaaga aga
239121DNAHomo sapiens 91cagaccaccc tcaaattgag t
219221DNAHomo sapiens 92ccagttgagc ctctgaatga c
219321DNAHomo sapiens
93gcagtcattc agaggctcaa c
219421DNAHomo sapiens 94cacagggcaa ggatacagag a
219521DNAHomo sapiens 95tcctcagctc acctctctgt a
219621DNAHomo sapiens
96agtgacagtc tgggacatct g
219721DNAHomo sapiens 97aggattcaga tgtcccagac t
219821DNAHomo sapiens 98aagatgatcc ccaacaatgc t
219920DNAHomo sapiens
99caggaatggg tggcatatgc
2010021DNAHomo sapiens 100tctgagcaca caatccactc t
2110121DNAHomo sapiens 101aggaagtcca ctatgcttgc t
2110222DNAHomo sapiens
102ccttgattca caatggctca gt
2210321DNAHomo sapiens 103gcactgagcc attgtgaatc a
2110421DNAHomo sapiens 104ctggtttgtg gaggagagag t
2110520DNAHomo sapiens
105ttggattcca ggggcactct
2010621DNAHomo sapiens 106gccactcaca ttgttgctga a
2110721DNAHomo sapiens 107gatcctgctg ttcagcaaca a
2110821DNAHomo sapiens
108actgcaaacc aaaaggtgct t
2110920DNAHomo sapiens 109ccggtcactt cacgatgaca
2011022DNAHomo sapiens 110aacttctcca agcacaaagt gt
2211121DNAHomo sapiens
111cggcttcagg aattcacaga a
2111221DNAHomo sapiens 112cctaggttgt ctccactgct a
2111321DNAHomo sapiens 113ggaggtcctg tgtatgagga t
2111421DNAHomo sapiens
114actgctgaac acacttttcc t
2111521DNAHomo sapiens 115gagccctaca gcagtgaaaa g
2111621DNAHomo sapiens 116acctgggcat ttaccttcag a
2111723DNAHomo sapiens
117gaactagggt agaggcacta gag
2311823DNAHomo sapiens 118ccccctttcc tgactactta act
2311922DNAHomo sapiens 119tcacctgacc tctgtgttta ca
2212021DNAHomo sapiens
120gcatattcat ggcagctggt a
2112121DNAHomo sapiens 121agtccccaaa aacaggtgga a
2112223DNAHomo sapiens 122acaggcatgt tttctttcct aga
2312323DNAHomo sapiens
123gctgctggct agtgataaat aac
2312421DNAHomo sapiens 124ctctgtgccc atctctacca t
2112522DNAHomo sapiens 125agcttagaca aagatgggca aa
2212620DNAHomo sapiens
126cattccccgg cttgtaaagg
2012721DNAHomo sapiens 127cacaaacaga aggccaagtg a
2112821DNAHomo sapiens 128aaaggtgatg aagggccagt t
2112921DNAHomo sapiens
129ctccgtcaga gtctccagaa g
2113022DNAHomo sapiens 130cgttttctgt ggcaaccata ac
2213123DNAHomo sapiens 131cagaactggg caagaaaatg ttt
2313221DNAHomo sapiens
132gttgtccatc gagttgtcat g
211332714DNAMus musculus 133cgggttccgg aaagcgggtg gcgcaggcgg ggagcgcgtc
agggagcggc cctggctgcg 60gggtcgcggc cttggagaga tgatgcgggg cgcaggacga
ttcacgatcc caggtggact 120tgcgcgctcg aaaccagcga tcacagggcc caaatggaaa
ccagagagct ccgaataggc 180cacagcggtc agttacccag gactgcagag acacccttca
ctcctgcatg cgtttgatct 240gactccacca gaaggtccag tcgccaacgc ctagctgagg
tcccaggcca ggccttaccc 300ccaaccccta ccctctcagc ctccaattga aagaaggcct
gccggctcca actcaattag 360cacaaaggcg gcggcgggca ccctggtctg tgccgcaggg
cagggagcgt cctggcgggc 420aggtagagag aagtccgcta gccccgggtg ttatcagcaa
gcggcttaat caggaatttg 480agctgacaac gtgcgcaccg ccacccggct ccgccagccg
gttgacggtg ggagtgagac 540ccgagaggct ggagtcccag acttcaaagc tgcccgaggt
gcggcggcct tggggaaggg 600ggcgcccggc gacaatcggc aaccccgagc gccgcctgga
cagaagccgg cgggccagtg 660ggcgctggac catcttccct gcgaatacaa tgcagctgtc
ttcagacaca cactagaaga 720gtgcagtgga ctccattaca gatggctgtg agccaccatg
tggttgctgg taattgaact 780caggacctct ggaagagcaa tctgtgctct taactgctga
gccacctctc cagttcccac 840caccaccatg cccgctcggc cccccagaca tcacacaaag
aagaaaacga caaggcagac 900caacagatga acaggatcca gagtgaccag aaccagacca
gatgtcccat gatgacatca 960caggagggac cgacattgtg ggaatggtgc ttgaagatga
tggtgacact ttctgggaag 1020accttcccgg atctccagag gtccaaattt gcatggtgag
tcccaacctc caccactggt 1080ctttgtgccc tcctggtaga gctgtgccct gcccgtgtgg
ttataatggg gccatcacat 1140gtgtgtagag gatgaagaca gccacagtga catgccggac
atttattatg catcagtagt 1200ctccatccac tgtgcccaca gcacccactt gagagtcact
cctagctgac atggtacctc 1260aactggaatc ctctatccaa ggatcttgtg gaccacatga
ggctaattag aatatgctct 1320tctctgcggc tgctggtcag ggatgagcgc tctcccatgt
ctgcttctgt tagccgtgaa 1380tgagtctgcc ttctttttaa ggaatgaata tatattcttt
gatggttcca tacatgtaca 1440catgtactgt ctgttgatta tattttacgc cacacccctt
actcccccat gtcttcccaa 1500aatctcttct ctctcctctc ttctccctcc cctccatcct
cttccttctc ctcctcctct 1560tctttaaaac cacacccagt ccagtgagtg ttgcctgaac
acacatgggc attgagccat 1620cccagggact tgggcaacct accagcagtc atccatacct
ttggagaagc tcggccgtcc 1680ttccttggaa gcatccattg gcctatgacc tccggaccca
cagcttttga ctaggtgcat 1740ggcactagat ttgtgttccc tcctctgaag cagacctcag
atcccgtcag aaaacgggtg 1800gctcccacaa ccaccatgcc tctatggcac tggtagcagg
gcttgctttg gctttgttcc 1860ttaacttcca gctaccttga gatcttctag gcagctcccc
tgcagccaga ggaccagcta 1920ttcagctgcg tgtgacagat gacaatgttc cttcccctac
ccgcctgccc acccagagct 1980ctgacctggt gtcagcctct tgtgctgaat ggaggcatct
acagctgctg ggtgagtact 2040ctgaattccc tgatgtcaaa atgacgctcg ggaatgtgcc
agcctcatga gtctgttggt 2100ctgtctcact cattcactcc atcagctgag ggtgggatct
gaacagatcc ggttatgcat 2160catgtatatc actgggatgt gtaccgagag tcaccatgga
aacctgggta tgggactcag 2220agtcaccatg gaaaccagcc actgggtatg cctgtcagga
agcttctaga gtgtgttcac 2280ggaagtggga agagccaccc tgcgtgttgg cagtgccatc
tcttcgtgat ctggggtcct 2340ggacagaatg aaaaggagca agcaagtgga gtaccagcag
tcacccctct ctgcttcctg 2400actgtgggtg ccatgtgacc agctgtttca tgttcctgcc
actgtgcctc cccgccatga 2460cggactgtgc acgctcacac tgtgagccag cgcgaaccct
tccttcttta aggtgctttt 2520tgttacagcg gtgagacgac ggctaatatg ctgcagaaac
acgtgcacgg tgtgcagggc 2580tcacgtggac gacacaatcc ctgctgcatc tcatttgact
agctctcagc atctgcttca 2640gccgctcttc taggaaacca ctcttattct tctggactct
cttttgctca taaattaaaa 2700tgcaaattct atca
27141342458DNAMus musculus 134cgggttccgg aaagcgggtg
gcgcaggcgg ggagcgcgtc agggagcggc cctggctgcg 60gggtcgcggc cttggagaga
tgatgcgggg cgcaggacga ttcacgatcc caggtggact 120tgcgcgctcg aaaccagcga
tcacagggcc caaatggaaa ccagagagct ccgaataggc 180cacagcggtc agttacccag
gactgcagag acacccttca ctcctgcatg cgtttgatct 240gactccacca gaaggtccag
tcgccaacgc ctagctgagg tcccaggcca ggccttaccc 300ccaaccccta ccctctcagc
ctccaattga aagaaggcct gccggctcca actcaattag 360cacaaaggcg gcggcgggca
ccctggtctg tgccgcaggg cagggagcgt cctggcgggc 420aggtagagag aagtccgcta
gccccgggtg ttatcagcaa gcggcttaat caggaatttg 480agctgacaac gtgcgcaccg
ccacccggct ccgccagccg gttgacggtg ggagtgagac 540ccgagaggct ggagtcccag
acttcaaagc tgcccgaggt gcggcggcct tggggaaggg 600ggcgcccggc gacaatcggc
aaccccgagc gccgcctgga cagaagccgg cgggccagtg 660ggcgctggac catcttccct
gcgaaacatc acacaaagaa gaaaacgaca aggcagacca 720acagatgaac aggatccaga
gtgaccagaa ccagaccaga tgtcccatga tgacatcaca 780ggagggaccg acattgtggg
aatggtgctt gaagatgatg gtgacacttt ctgggaagac 840cttcccggat ctccagaggt
acggaaccga ggtccaaatt tgcatggtga gtcccaacct 900ccaccactgg tctttgtgcc
ctcctggtag agctgtgccc tgcccgtgtg gttataatgg 960ggccatcaca tgtgtgtaga
ggatgaagac agccacagtg acatgccgga catttattat 1020gcatcagtag tctccatcca
ctgtgcccac agcacccact tgagagtcac tcctagctga 1080catggtacct caactggaat
cctctatcca aggatcttgt ggaccacatg aggctaatta 1140gaatatgctc ttctctgcgg
ctgctggtca gggatgagcg ctctcccatg tctgcttctg 1200ttagccgtga atgagtctgc
cttcttttta aggaatgaat atatattctt tgatggttcc 1260atacatgtac acatgtactg
tctgttgatt atattttacg ccacacccct tactccccca 1320tgtcttccca aaatctcttc
tctctcctct cttctccctc ccctccatcc tcttccttct 1380cctcctcctc ttctttaaaa
ccacacccag tccagtgagt gttgcctgaa cacacatggg 1440cattgagcca tcccagggac
ttgggcaacc taccagcagt catccatacc tttggagaag 1500ctcggccgtc cttccttgga
agcatccatt gtaagtagat cttcacccag ggatgggatt 1560ttatgagcct ctcccgtcca
tgctggatat tgactgactc catcgtgtgt gaatcaccac 1620agctgctgtg agttcgtgaa
tccgatgctc acacatcgtg gccagaggac agcatttcac 1680tcccctctcc cagcctctag
ctttgatgtt ctttctgctt cctctcccat aatgtcccct 1740gagcttcaca ggggataggt
ggatataggt gtcccatcag gggtgaacac tcacggtcac 1800ttactgttag cactttgatc
aagccgtgag tcccagcact aaccactaca ctggaagaaa 1860cagcttcctg agcaatgcta
atttatggat ataaatagaa atattgagaa ggcaatttga 1920cagcgtgtcc atttaaccgg
tagtttctcc cctagggcct atgacctccg gacccacagc 1980ttttgactag gtgcatggca
ctagatttgt gttccctcct ctgaagcaga cctcagatcc 2040cgtcagaaaa cgggtggctc
ccacaaccac catgcctcta tggcactggt agcagggctt 2100gctttggctt tgttccttaa
cttccagcta ccttgagatc ttctaggcag ctcccctgca 2160gccagaggac cagctattca
gctgcgtgtg acagatgaca atgttccttc ccctacccgc 2220ctgcccaccc agagctctga
cctggtgtca gcctcttgtg ctgaatggag gcatctacag 2280ctgctggaaa ttagagcgct
ccgttccgtg gtattacgcg gcggcactcc acgggcctgg 2340gagtgcagag ctgattggat
gtcccaccca aaggagagac ggttcaagct ctttccagga 2400aattgagtgg aacacccaaa
ctgacatcca atattaaata ttgtatttgt ggacttgt 245813528RNAHomo sapiens
135ggcuaccuuc ugcaugcauu ucuguugg
2813622RNAHomo sapiens 136acagcaggca cagacaggca gu
2213715RNAHomo sapiens 137auccgagccc ugcug
1513839RNAHomo sapiens
138aauugcccac acuucacccu aaagacagau ccugcuguu
3913916RNAHomo sapiens 139uccugcaacc ugcugc
1614046RNAHomo sapiens 140agcuugggug accauacauu
cuaggaaaga aaacaugccu guuguc 4614128RNAHomo sapiens
141aacugggcaa gaaaauguuu cugcuguu
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