Validation of the novel lincRNAs.

<p>(A) 26 randomly selected novel lincRNAs from the final lincRNA list were subjected to RT-PCR validations, among which 8 were divergent lincRNAs (the transcript id is marked in red color and ends with a ‘*’ suffix). The PCR products were visualized on Agoras gel and the sizes of DNA markers (M) are shown on the right. (B) Comparison of the identified novel lincRNAs with NONCODE v3.0. 299 transcripts (32.6%) were found in common.</p

Sebnif: An Integrated Bioinformatics Pipeline for the Identification of Novel Large Intergenic Noncoding RNAs (lincRNAs) - Application in Human Skeletal Muscle Cells

<div><p><i>Ab initio</i> assembly of transcriptome sequencing data has been widely used to identify large intergenic non-coding RNAs (lincRNAs), a novel class of gene regulators involved in many biological processes. To differentiate real lincRNA transcripts from thousands of assembly artifacts, a series of filtering steps such as filters of transcript length, expression level and coding potential, need to be applied. However, an easy-to-use and publicly available bioinformatics pipeline that integrates these filters is not yet available. Hence, we implemented sebnif, an integrative bioinformatics pipeline to facilitate the discovery of <i>bona fide</i> novel lincRNAs that are suitable for further functional characterization. Specifically, sebnif is the only pipeline that implements an algorithm for identifying high-quality single-exonic lincRNAs that were often omitted in many studies. To demonstrate the usage of sebnif, we applied it on a real biological RNA-seq dataset from Human Skeletal Muscle Cells (HSkMC) and built a novel lincRNA catalog containing 917 highly reliable lincRNAs. Sebnif is available at <a href="http://sunlab.lihs.cuhk.edu.hk/sebnif/" target="_blank">http://sunlab.lihs.cuhk.edu.hk/sebnif/</a>.</p></div

Analysis of the novel lincRNAs in HSkMC.

<p>(A) Cumulative curve of the average PhastCons score of the novel lincRNAs (green) compared to randomly selected genome background (blue) and known mRNAs (red). These novel lincRNAs are more conserved than the genome background but less conserved than the mRNAs. (B) Comparison of expression profiles of novel lincRNAs (green), known ncRNAs (blue) and known mRNAs (red). Both novel lincRNAs and the known ncRNAs are expressed at a lower level than known mRNAs. (C) 57% (523 out of 917) of the novel lincRNAs are divergent transcripts generated within 2 kbp upstream of known protein coding genes. (D) Gene Ontology annotation of the above protein coding genes. The y-axis shows the top 10 enriched GO terms and the x-axis shows the enrichment significance P-values.</p

Identification of novel lincRNA catalog in HSkMC.

<p>(A) The raw RNA-seq data was pre-processed, aligned with Tophat and assembled using Cufflinks in <i>ab initio</i> mode. (B) Sebnif filtering on the assembled transcripts. The numbers in parentheses represent the number of transcripts after each filtering step. (C) Annotating and further filtering of the novel lincRNAs with H3K4me3 and CAGE data.</p

Snapshots of sebnif web server.

<p>(A) The data upload page. All the parameters of sebnif could be specified by the users through this page. (B) The result page showing the report of novel lincRNAs identified in Human Skeletal Muscle Cells. The final list of novel lincRNAs in standard GFF format and the iSeeRNA noncoding score for each transcript can be downloaded directly; statistic numbers during the filtering steps and the FRFE Profile and STGE Profile generated by FRFE and STGE algorithms were also provided for users to evaluate the quality of the data.</p

miR-29 is anti-fibrogenic in C2C12 cells.

<p>(A) C2C12 cells were differentiated (DM) for 0, 1, 2 or 4 days, at which times total RNAs were isolated for qRT-PCR measurement of the expressions of Col 1A1, Col 1A2, Col 3A1, α-SMA or VIM as well as MyHC, α-Actin, Troponin and MyoG. Expression folds are shown with respect to 0 hr cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (B and C) C2C12 cells were transfected with negative control (NC) or miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Myogenin, Troponin, α-Actin or MyHC as well as Col 1A1, Col 1A2, Col 3A1, α-SMA or VIM. Expression folds are shown with respect to NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (D) C2C12 cells were transfected with negative control (Anti-NC) or Anti-miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Col 1A1, Col 1A2, and Col 3A1. Expression folds are shown with respect to Anti-NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (E) Wild type (WT) or Mutant Col 1A1, Col 1A2, or Col 3A1-3′UTR luciferase reporter constructs were transfected into C2C12 cells with indicated miRNA or negative control (NC) oligos. Luciferase activities were determined at 48 h post-transfection and normalized to β-Galactosidase protein. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represent the average of three independent experiments ± S.D. (F) A schematic illustration of base pairing between mmu-miR-29c with 3078–3099 region on 3′UTR of mouse Lims1. (G) Lims1 protein expression was measured in NC or miR-29 stable C2C12 cells by Western blotting using GAPDH as a loading control. (H) NC or miR-29 stable cells were differentiated and Lims1 expression levels were measured at the indicated time points. (I) C2C12 cells were transfected with negative control (Anti-NC) or Anti-miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Lims1. Expression folds are shown with respect to Anti-NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (J) Wild type (WT) or Mutant Lims1-3′UTR luciferase reporter constructs were transfected into C2C12 cells with miR-29 or negative control (NC) oligos. Luciferase activities were determined at 48 h post-transfection and normalized to β-Galactosidase protein.</p

Smad3 suppresses miR-29 promoter through inhibiting MyoD binding and enhancing YY1 recruitment.

<p>(A) Schematic illustration of proximal promoter region of mmu-miR-29b/c primary transcript. The arrow denotes the Transcriptional Start Site (TSS). Predicted Smad3 (S), MyoD (M) and YY1 (Y) binding sites were displayed. The location of each site was indicated below. (B) C2C12 myoblasts were untreated or treated with TGF-β for 12 hrs at which time chromatins were collected for ChIP assays using antibodies against MyoD or IgG as controls. PCR assays were then used to measure the enrichment fold of MyoD on four putative binding sites, M1/M2, M3 and M4. (C, D and E) ChIP-PCR assays were performed as above to examine the binding of YY1, Ezh2 and H3K27me3 to putative YY1 binding sites, Y1, Y2, Y3 or Y6. Enrichment folds are shown with respect to IgG control where normalized PCR values were set to 1. Data are plotted as mean ± S.D. (F) Upper: 10T1/2 cells were transfected with 0.25 µg of miR-29-promoter-luc reporter plasmid along with 0.5 µg YY1 plasmid and Smad3 plasmid (0, 0.20, 0.50, 1.00, or 2.00 µg). 24 hr post-transfection, cells were treated with TGF-β for 48 hrs at which time luciferase activities were determined. (G) The transfections were performed as above with 0.5 µg of MyoD plasmid and Smad3 expression plasmid (0, 0.20, 0.50, 1.00, or 2.00 µg). (H) The transfections were performed as above with indicated plasmids (0.5 µg of MyoD, YY1 or Smad3 plasmids were used). (I) C2C12 cells were transfected with siRNA oligos knocking down Smad3, MyoD or YY1. The expression of Lims1 was examined by Western blotting using Tubulin as a loading control. *p<0.05. ** p<0.01.</p

A model of TGF-β-Smad3-miR-29 circuit in myogenic and fibrogenic differentiation of C2C12 myoblasts.

<p>The model depicts the roles of the TGF-β-Smad3-miR-29 regulatory circuit in myogenic and fibrogenic differentiation of C2C12 cells. In the normal myogenesis, the recruitment of MyoD/SRF and the displacement of YY1/PRC from miR-29 promoter lead to the elevation of miR-29 expression and its feedback inhibition on YY1 and successful myogenic differentiation. Upon TGF-β stimulation, activated Smad3 translocates into nucleus where it binds to SBE, resulting in MyoD dissociation as well as YY1/Ezh2 recruitment to multiple CCAT boxes of miR-29b/c promoter. This leads to trimethylation of histone lysine 27 and subsequent silencing of miR-29 expression. Loss of miR-29 upregulates the expression of ECM genes such as Collagens as well as cell adhesion genes such as Lims1, thus promoting the conversion of myoblasts into myofibroblasts. Straight line, promoter/enhancer region of mmu-miR-29b/c with arrow denotes TSS; CCAT, YY1 binding elements; E-box, MyoD binding sites; SBE, Smad3 binding element; Me, methylation of histone lysine 27; Ac, acetylation of histones.</p

Smad3 mediates the repression of TGF-β on miR-29 at the transcriptional level by binding to miR-29 promoter region.

<p>(A) C2C12 myoblasts were transfected with siRNA oligos against Smad2, Smad3 or Smad7, using scrambled siRNAs (SCR) as a control. 48 hr post-transfection, the expressions were examined by Western blotting using α-Tubulin as a loading control. (B) C2C12 myoblasts, transfected with the indicated siRNA oligos, were incubated with TGF-β in DM for 48 hrs at which time the expressions of miR-29 were measured. (C) miR-29-promoter-luc reporter activities in C2C12 myoblasts transfected with the above siRNA oligos and then treated with TGF-β in DM for 48 hrs. (D) miR-29 expressions in primary myoblasts isolated from <i>Smad3^+/+</i>, <i>Smad3^+/−</i> or <i>Smad3^−/−</i> mice. (E) miR-29 expressions in primary myoblasts from <i>Smad7^+/+</i> or <i>Smad7^−/−</i> mice. (F) <i>Smad7^+/+</i> or <i>Smad7^−/−</i> mice were injected with Cardiotoxin (CTX) into TA muscles to induce muscle regeneration. Muscles were harvested at designated days after the injection and assayed for miR-29 expression levels by qRT-PCR. (G) C2C12 myoblasts were untreated or treated with TGF-β for 12 hrs at which time chromatins were collected for ChIP assays using antibodies against Smad3 or IgG as controls. PCR assays were then used to measure the enrichment fold of Smad3 on three putative binding sites, S1, S2 and S3.</p

TGF-β inhibits miR-29 during myogenic and fibrogenic differentiation of C2C12 cells.

<p>(A) IF staining of MyoD (red) and α-SMA (green) in C2C12 cells treated with or without TGF-β. Photos were taken by confocal scanning microscope. Dashed oval is used to circle α-SMA+/MyoD− cells; arrow, α-SMA+/MyoD+ cell. Scale bar = 50 µm. (B) miR-29 expressions in C2C12 incubated without (−) or with (+) TGF-β for 0, 3 or 6 days in DM. (C) miR-29 promoter luciferease reporter activities in C2C12 incubated with indicated doses of TGF-β for 48 hrs in DM. (D) C2C12 cells stably expressing miR-29 or vector control (NC) were treated with TGF-β in DM for 0, 2, 4, or 6 days at which times MyHC, Troponin, α-Actin were probed by Western blotting. (E) Cells were photographed under phase contrast or immunostained for MyHC and visualized by confocal scanning microscope at DM day 4. Positively stained cells were quantified. Data are plotted as mean ± S.D. Scale bars = 50 µm. (F) NC or miR-29 stable cells were treated without or with TGF-β for 48 hrs at which time Col 1, α-SMA and Lims1 were probed by Western blotting with α-Tubulin as a loading control. (G) Col 1, α-SMA, VIM and Lims1 were stained by IF and visualized by confocal scanning microscope. Scale bars = 50 µm. (H) mRNA expressions of Col 1A1, Col 3A1, α-SMA and Lims1. Data are plotted as mean ± S.D. *p<0.05.</p