Journal: Genes & Development

Article Title: The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration

doi: 10.1101/gad.234419.113

Figure Lengend Snippet: H19 promotes muscle differentiation in C2C12 cells, primary human satellite cells, and primary mouse myoblasts; its trans -regulatory function is mediated by miR-675-3p and miR-675-5p. We transfected siRNAs specific to mouse or human H19 in C2C12 myoblast cells or primary human satellite cells in GM and changed to DM. The next day (DM1), siRNAs were again transfected, and the cells were harvested for RNA analysis on DM3. Alternatively, we transfected cells with GL2 (22 bases from luciferase gene) as negative controls. Knockdown of H19 decreases H19 , miR-675-3p, and miR-675-5p levels in C2C12 myoblast cells ( A ) and human satellite cells ( C ) and results in decreased expression of the differentiation markers myogenin and MHC mRNAs ( B , D ). ( B , D ) Exogenous miR-675-3p and miR-675-5p rescue the differentiation in both cell types. ( E , F ) Satellite cells derived from H19 -deficient mice ( H19 ΔME/+) lack H19 , miR-675-3p, and miR-675-5p ( E ) and show decreased expression of myogenin and MHC mRNAs at DM1 ( F ). ( F ) Exogenous miR-675-3p and miR-675-5p restore expression of the differentiation markers. The normalization for H19 , myogenin, MHC mRNAs, and microRNAs was as described in . Mean ± SEM of three biological replicates are shown. (*) P < 0.001; (**) P < 0.005.

Article Snippet: A pool of three siRNAs against mouse Cdc6 was purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Luciferase, Knockdown, Expressing, Derivative Assay

Journal: Genes & Development

Article Title: The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration

doi: 10.1101/gad.234419.113

Figure Lengend Snippet: Smad1, Smad5, and Cdc6 are important targets for miR-675-3p and miR-675-5p. ( A–C ) Cotransfection of miR-675-3p or miR-675-5p microRNA into U2OS osteosarcoma cells repressed Renilla (rr) luciferase activity when the rr reporter was fused to the target 3′ UTR. Mutations in the target sites in the 3′ UTR (Supplemental Fig. 7) relieved the repression. A firefly (pp) luciferase plasmid was cotransfected as a transfection control. The rr/pp was normalized to that for a control Renilla luciferase plasmid with a vector 3′ UTR (PRL) and expressed relative to the normalized rr/pp in cells transfected with the GL2 RNA control. Mean ± SEM of three individual experiments. (*) P < 0.001. ( D–F ) Transfection of miR-675-3p or miR-675-5p in C2C12 myoblast cells in GM decreased Smad1, Smad5, and Cdc6 protein levels as detected by Western blotting 2 d after the second transfection. GL2 served as a control oligonucleotide. β-Actin or GAPDH was used as a loading control. ( G–I ) 2′ O -methyl oligonucleotide against miR-675-3p or LNA antisense oligonucleotide against miR-675-5p (anti-675-3p or anti--675-5p) increased endogenous Smad1, Smad5, and Cdc6 protein levels in C2C12 cells cultured in DM for 2 d. 2′ O -methyl or LNA antisense GL2 (anti-GL2) served as a control oligonucleotide. β-Actin or GAPDH is used as a loading control. ( J–L ) As measured by qRT–PCR, miR-675-3p or miR-675-5p target genes display anti-correlation in their temporal expression pattern during regeneration of skeletal muscle after CTX injury. ( M ) C2C12 myoblast cells were transfected with GL2 control siRNA, H19 siRNA, or H19 siRNA combined with siRNAs specific to Smad1, Smad5, and Cdc6 once in GM and then on DM1, and cells were harvested on DM3. qRT–PCR was performed for myogenin and MHC. The normalization for myogenin and MHC mRNAs was as described in . Mean ± SEM of three biological replicates are shown. (*) P < 0.001; (**) P < 0.005.

Article Snippet: A pool of three siRNAs against mouse Cdc6 was purchased from Santa Cruz Biotechnology.

Techniques: Cotransfection, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Control, Western Blot, Cell Culture, Quantitative RT-PCR, Expressing