RNA interference affects the feak-headbox 3a gene expression in myoblast cell line L6

Ding Jie; Liang Bing-sheng; Da Zhi-feng; Zhu Zhi-xiang; Wei Jian; Jia Ying-wei; Feng Yong

doi:10.3969/j.issn.2095-4344.2013.33.012

Abstract： BACKGROUND:Recent studies found that some factors play important role in the process of denervated muscle atrophy, especial y the feak-headbox transcription factor, is the key element to regulate the denervated muscle atrophy. OBJECTIVE:To investigate the effect of RNA interference on inhibiting feak-headbox 3a gene expression in vitro. METHODS:The myoblast cel line L6 were cultured in the 6-wel cel culture plates, then pEGFP-N1 and smal interfering RNA recombinant plasmid with the same ratio was transfected under the Lipofectamine2000 mediation to optimize the transfection efficiency of the detection system;2μg smal interfering RNA recombinant plasmid of feak-headbox 3a gene were transfected with myoblast cel line L6 for 48 and 72 hours. RESULTS AND CONCLUSION:At 48 hours after pEGFP-N1 and siRNA recombinant plasmid transfection, a large number of bright green fluorescent displayed under fluorescence microscope with higher transfection efficiency. Real-time quantitative PCR analysis showed that there were significant differences in the sequences of feak-headbox 3a-Ⅰ, feak-headbox 3a-Ⅱ, feak-headbox 3a-Ⅲ, feak-headbox 3a-Ⅳ on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after trasfection (P<0.05), and the inhibition effect was more significant at 72 hours after transfection when compared with that at 48 hours after transfection. Western Blot gray analysis showed that there were significant differences in sequences of feak-headbox 3a-Ⅰ, feak-headbox 3a-Ⅱ, feak-headbox 3a-Ⅲ, feak-headbox 3a-Ⅳ on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after trasfection (P<0.05), and the inhibition effect was more significant at 72 hours after transfection when compared with that at 48 hours after transfection, which was same with the effect on mRNA level. RNA interference in vitro can significantly inhibit the fork-head transcription factor feak-headbox 3a gene expression, and the inhibition effect of feak-headbox 3a gene smal interfering RNA recombinant plasmid transfected with the sequence on the mRNA and protein level of feak-headbox 3a is not clear, which can provide new idea for the gene therapy of RNA mediated denervated skeletal muscle atrophy.