干扰Mtmr 3基因对C2C12细胞增殖与分化的影响

Effect of Interfering Mtmr 3 on C2C12 Cells Proliferation and Differentiation

旨在探究肌管相关蛋白3(myotubularin related protein 3,Mtmr 3)对C2C12细胞增殖与分化的调控作用及机制。本试验以小鼠成肌细胞系(C2C12)为试验材料,分别使用qRT-PCR和免疫荧光染色检测Mtmr 3基因在成肌细胞生长期与分化期的mRNA表达水平和分化第6天时的分布形态。合成Mtmr 3的小干扰RNA(small interfering RNA,siRNA),试验分为siRNA NC对照组和Mtmr 3 siRNA处理组(n=3),利用EdU、CCK-8、qRT-PCR、Western blot技术检测Mtmr 3 siRNA对成肌细胞增殖与分化的影响,并通过信号通路研究调控成肌细胞增殖与分化的机制。结果显示,Mtmr 3在生长期的mRNA水平表达量总体呈下降趋势,而在分化期的表达量呈逐渐上升趋势,Mtmr3免疫荧光染色呈肌管状。干扰Mtmr 3后,细胞内Mtmr 3基因的mRNA和蛋白表达水平均极显著地降低(P<0.01);在增殖试验中,转染细胞Mtmr 3 siRNA后,极显著地增加了EdU阳性细胞占总细胞的比率和细胞活力(P<0.01);干扰Mtmr 3表达后,Pcna和Cdk4的mRNA与蛋白表达水平极显著地升高(P<0.01),Ccnd基因的mRNA表达水平极显著地升高(P<0.01)。在分化试验中,转染Mtmr 3 siRNA后极显著抑制了分化标志基因Myhc蛋白的表达水平(P<0.01),极显著抑制细胞分化第4和6天Mtmr 3、Myog和Myhc基因在mRNA的表达水平(P<0.01)。在增殖期和分化期干扰Mtmr 3表达后,Mtor和P70s6k的磷酸化蛋白表达水平分别极显著升高和极显著降低(P<0.01)。CCK-8的结果表明,与对照组相比,Mtmr 3 siRNA能够抵消部分Mtor通路特异性抑制剂Rapamycin(Rapa)的抑制作用,促进成肌细胞增殖(P<0.01);在细胞分化时加入Rapa处理成肌细胞,细胞内Mtor、P 70s6k、Myog和Myhc基因的mRNA表达水平均显著降低(P<0.05)。综上所述,Mtmr 3在成肌细胞生长期的表达量呈下降趋势,而在分化期的表达量呈逐渐上升趋势,Mtmr3免疫荧光染色呈肌管状。干扰Mtmr 3基因表达,通过激活Mtor通路促进成肌细胞增殖,通过失活Mtor通路抑制成肌细胞分化。

This study aimed to investigate the regulation and mechanism of Mtmr 3(myotubularin related protein 3)on proliferation and differentiation of C2C12 cells.In this study,mouse myoblast cell line(C2C12)was used as experimental material.qRT-PCR and immunofluorescence staining were used to detect the mRNA expression level of Mtmr 3 gene in myoblast growth and differentiation stages,and the distribution morphology on day 6 of differentiation.The small interfering RNA(siRNA)of Mtmr 3 was synthesized and divided into siRNA NC control group and Mtmr 3 siRNA treatment group(n=3).EdU,CCK-8,qRT-PCR and Western blot were used to detect the influence of Mtmr 3 siRNA on myoblast proliferation and differentiation,and the relevant mechanisms of regulating myoblast proliferation and differentiation were studied through signal pathways.The results showed that the mRNA expression level of Mtmr3 decreased in the growth stage,but increased gradually in the differentiation stage,and the immunofluorescence staining of Mtmr3 showed a myotube shape.After interfering with Mtmr 3,mRNA and protein expression levels of Mtmr3 gene were significantly decreased(P<0.01).In proliferation test,the percentage of EdU positive cells in total cells and cell viability were significantly increased after transfection of Mtmr3 siRNA(P<0.01).After the interference of Mtmr3 expression,the mRNA and protein expression levels of Pcna and Cdk4 were significantly increased(P<0.01),and the mRNA expression level of Ccnd gene was significantly increased(P<0.01).In the differentiation test,the expression level of differentiation marker gene Myhc protein was significantly inhibited after transfection of Mtmr3 siRNA(P<0.01),and the mRNA expression levels of Mtmr3,Myog and Myhc genes were significantly inhibited at the 4th and 6th day of cell differentiation(P<0.01).After interfering Mtmr 3 expression in proliferative stage and differentiation stage,phosphorylated protein expression levels of Mtor and P70s6k were significantly increased and significantly decreased,respectively(P<0.01).The results of CCK-8 showed that Mtmr3 siRNA could counteract the inhibitory effect of Rapamycin(Rapa),a specific inhibitor of Mtor pathway,and promote myoblast proliferation compared with the control group(P<0.01);The mRNA expression levels of Mtor,P70s6k,Myog and Myhc genes were significantly decreased in myoblasts treated with Rapa during cell differentiation(P<0.01).In summary,the expression level of Mtmr3 in myoblast growth stage showed a downward trend,while that in differentiation stage showed a gradual upward trend,and the immunofluorescence staining of Mtmr3 showed a myotube shape.Interfering with Mtmr3 expression promotes myoblast proliferation by activating the Mtor pathway,and inhibits myoblast differentiation by inactivating the Mtor pathway.