| MicroRNAs (miRNAs) are a new class of non-coding RNAs which regulate gene expression at post-transcription level and play roles in development, tumorigenesis, embryonic stem cell differentiation apoptosis etc. Many miRNAs have been found to participate in skeletal muscle development in recent years. In our previous study, we analyzed miRNA expression profiles in porcine fetal and adult longesimmus muscle. MiR-214 and miR-495 was found to be highly expressed in embryonic skeletal muscle at 33 and 65 days post gestation, but were barely detected in adult skeletal muscle. MiR-133b had high expression level in both fetal and adult longesimmus muscle. Up to now, the funtion of miR-133b, miR-214 and miR-495 in myogenesis was not very clear. In this study, we try to explore the role of miR-133b, miR-214 and miR-495 in myogenesis and identify the new targets of these miRNAs.The myoblast cell line C2C12 was used for functional analysis of miR-133b, miR-214 and miR-495 in vitro. The results are as follows:(1) The expression of miR-133b, miR-214 and miR-495 in C2C12 cells cultured in growth medium (Od) and differentiation medium (DM) for 1 day (1d),2 days (2d), or 4 days (4d) was analized by Q-PCR. The results showed miR-133b, miR-214 and miR-495 were up-regulated during differentiation. The expression level of miR-133b and miR-495 at 1d was higher than Od, while the expression level of miR-214 at 1d was the same as Od; the expression level of miR-133b at 4d was 25 fold higher than that of Od, the expression level of miR-495 at 4d was 7 fold higher than that of Od; the expression level of miR-214 at 4d was 2 fold higher than that of Od.(2) The percentage of cells in G1 phase significantly increased (p<0.05) and the percentage of cells in S phase significantly decreased (p<0.05) with miR-133 overexpression, while the percentage of cells in G1 phase significantly decreased (p<0.01) and the percentage of cells in S phase significantly increased (p<0.01) with miR-133 knockdown. These results suggested miR-133b plays role in repressing C2C12 cell proliferation. Differentiation of C2C12 cells was repressed by the miR-133b knockdown, as indicated by the significant decrease in MyHC and myogenin expression after inhibition of endogenous miR-133b (p<0.01).(3) We analyzed C2C12 cells proliferation after inhibition of endogenous miR-214 by xCELLigence system. The result showed the cell indexes of cells with miR-214 knockdown were consistently lower than the controls, which meaned the growth speed of miR-214 knockdown cells was slower than that of control.This suggested that proliferation of C2C12 cells was repressed with miR-214 knockdown. The percentage of cells in G1 phase significantly increased (p<0.05) and the percentage of cells in S phase and G2 phase significantly decreased (p<0.05) with miR-214 knockdown. The result was concordant with the real-time proliferation analysis result. Myogenesis was repressed by downregulation of endogenous miR-214, as indicated by the significant lower expression level of MyHC-2d and myogenin in cells with miR-214 knockdown (p<0.05).(4) Expression of myogenin was up-regulated by overexpression of miR-495 in C2C12 cells (p<0.01). But no significant difference in expression of the MyHC-2d and Myh7 between cells with miR-495 overexpression and control cell.(5) The targets of miR-133b predictied by TargetScan and PicTar are FGFR1, ARHGEF9, Ppp2ca, Ppp2cb; targets of miR-214 are FGFR1, MAPK8, CDGAP, ARHGEF9 and Ppp2cb; target of miR-495 is MAPK10. Through luciferase repoter analysis, we found miR-133b repressed 3 UTR of FGFR1、ARHGEF9、Ppp2ca and Ppp2cb. We found miR-133b repressed protein of FGFRland PP2AC (Ppp2ca and Ppp2cb) in C2C12 cells by western blot. There was no significant difference in expression of FGFR1 and PP2AC mRNA in C2C12 cells cultured in differentiation medium for Od, 1d,2d or 4d. But the protein of FGFR1 and PP2AC both was significantly down-regulated during C2C12 differentiation.(6) miR-133b can repress the activation of p-ERK1/2 in C2C12 cultured in differentiation medium, indicated by down-regulation of p-ERK1/2 expression in cells with miR-133b overexpression; Inhibition of p-ERK1/2 in C2C12 cells significantly up-regulated the expression of miR-133b (p<0.01).(7) Inhibition of p-ERK1/2 in C2C12 cells cultured in growth medium by PD98059 repressed proliferation of cells, and significantly up-regulated the expression of myogenin (p<0.01); inhibition of p-ERK1/2 in C2C12 cells cultured in differentiation medium promoted the expression of myogenin and MyHC-2d significantly 20h and 36h after adding PD98059, but there is no significant difference in expression of the MyHC-2d and myogenin between cells treated with PD98059 and DMSO(control) for 65h. |
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