The purpose of the study was to investigate the influence of RACK1genesilencing by RNA interference (RNAi) on the expressions of two myogenic differentiation markgenes (MHC and MyoG) in C2C12 myoblast.We transfected three chemically synthesized RACK1 siRNA (siRNA-1,2,3) into C2C 12 myoblasts,and detected the interference efficiency with RT-qPCR in order toselect thesiRNA with the highest interference efficiency.Then,we transfected the selected siRNA into C2C12 cells and induced C2C 12 differentiation.In order to study the effect of RACK1 gene silencing on the expressions of MHC and MyoG,we examined MHC and MyoG mRNA and protein expressions by using RT-qPCR,Western blot and immunofluorescence methods.In addition,the effect of the RACK1 silencing on the activation of pI3K/Akt and Erk/MApK pathway was detected by Western blot analysis.The result showed that RACK1-siRNA-2 had the highest interference efficiency,and its interference efficiency was approximately 70％ at 48 h after the transfection.C2C12 myoblasts were transfected with siRNA-2 and induced into differentiation for 48 h and 72 h respectively.RT-qPCR showed that MHC and MyoG mRNA were significantly decreased in siRNA-2 transfected cells after 48 h of differentiation;Western blot and immunofluorescence showed that MHC and MyoG protein were decreased after 72 h of differentiation.However,no significant changes in phosphorylation levels of Akt and Erk were observed.We conclude that downregulation of RACK1 by RNAi significantly inhibits the expressions of MHC and MyoG,indicating that RACK1 is involved in positive regulation of C2C 12 differentiation.%本实验旨在研究RNA干扰(RNAi) RACK1基因对C2C12成肌细胞中肌分化标志基因MHC和MyoG表达的影响.将人工合成的靶向RAKC1基因的3条siRNA(1,2,3)转染C2C12细胞,用RT-qPCR方法检测并筛选干扰效率最高的1条siRNA.将筛选出的siRNA转染C2C12成肌细胞并诱导细胞分化,通过RT-qPCR、Western blot和免疫荧光方法在mRNA及蛋白水平检测RACK1基因沉默后对MHC和MyoG表达的影响.此外,Western blot方法检测RACK1基因沉默后对PI3K/Akt和Erk/MAPK通路激活的影响.结果表明:RACK1-siRNA-2干扰效率最高,转染48 h后抑制率可达70％.随后,C2C12细胞转染siRNA-2,诱导分化48h后RT-qPCR表明,MHC和MyoG的mRNA表达均显著性下调(P＜0.05);诱导分化72 h后,Western blot和免疫荧光结果表明MHC和MyoG的蛋白表达明显低于对照组,但AKT和Erk磷酸化水平未见明显变化.上述结果表明,干扰RACK1能显著抑制MHC和MyoG的表达,提示RACK1正向调控C2C12成肌细胞的分化.
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