The Effection Of Rnai To The FOXO3a Gene Expression In Myoblasts Cell Line L6

Objective To investigate the effect of inhibiting FOXO3a gene expression in vitro,to lay the foundation for the gene therapy for the RNAi-mediated denervated skeletalmuscle.Method L6rat myoblasts were used to culture the rat muscle cell line L6,pEGFP-N1and siRNA recombinant plasmid the ratio under theLipofectamine2000-mediated transfection, optimize transfection efficiency of thedetection system;2μgFOXO3a gene siRNA recombinant plasmid transfected L6,48h and72h after transfection, the inhibition of FOXO3a the mRNA using real-timequantitative PCR siRNA recombinant plasmid, using Western blotting to detect theinhibitory effects of siRNA recombinant plasmids FOXO3a protein levels.Results1pEGFP-N1siRNA recombinant plasmid48h after transfection under afluorescence microscope visible cells express a large number of bright greenfluorescent display system with higher transfection efficiency. Real-time quantitativePCR analysis showed that at48h after transfection, interfere with the sequenceFOXO3a-I, the FOXO3a-II, the FOXO3a-III, of FOXO3a-IV on FOXO3a, themRNA inhibition rates were54%,25%,22%,27%, compared with the controlgroup were significant differences (P <0.05), and72hours after transfection,interference sequence FOXO3a-I, the FOXO3a-II, the FOXO3a-III, the FOXO3a-IVinhibition rates of FOXO3a mRNA were81%,52%,46%,55%, and the controlgroup showed a significant difference (P <0.05), compared with48h, the inhibitoryeffect is more apparent. But the inhibitory effect of the four pairs of sequences thereare significant differences, the most obvious inhibitory effect is FOXO3a-Ⅰ,the grayscale analysis of the Western blotting showed that48h after transfection,interfere with the sequence FOXO3a-I, the FOXO3a-II, the FOXO3a-III, theFOXO3a-IV,protein inhibition rates were46%,12%,11%,23%, and Compared tothe control group, there were significant differences (P <0.05),72hours aftertransfection, FOXO3a-I,FOXO3a-Ⅱ FOXO3a-Ⅲ, FOXO3a-Ⅳ inhibition rate ofprotein expression were72%,48%,38%,44%, compared with the control group,there are significant differences (P <0.05), compared with48h, the inhibitory effectis more obviousl.Conclusion:1RNA interference in vitro can significantly inhibit the forkheadtranscription factor FOXO3a gene expression.2, In vitro experiments FOXO3a genesiRNA recombinant plasmid transfected interference sequence to suppress the effectof the mRNA and protein of FOXO3a is not clear, RNAi-mediated denervatedskeletal muscle atrophy gene therapy offers a new way of thinking.