METTL21C下调对小鼠成肌细胞分化的影响

Effect of Down-regulation of METTL21C on Differentiation of Mouse Myoblasts

为探究甲基转移酶样21C(methyltransferase like 21C,METTL21C)在小鼠成肌细胞分化过程中的功能,设计2组METTL21C的shRNA干扰序列,构建靶向METTL21C的干扰载体。用慢病毒法侵染小鼠C2C12细胞,筛选获得稳定干扰METTL21C的C2C12细胞株并检测干扰效率,经过20 mL/L马血清诱导细胞分化后用实时荧光定量PCR及Western blot技术检测成肌分化相关基因表达。结果表明,成功构建两组靶向METTL21C的慢病毒干扰载体,实时荧光定量PCR及Western blot结果显示干扰组细胞中METTL21C表达水平均极显著降低(P<0.01),shRNA1组干扰效率最高(73.6%)。干扰组细胞中成肌分化标志基因MyoG、MyoD、Myf5、MRF4表达水平均极显著降低,MEF2C表达水平下调更为明显(P<0.01)。慢肌标志基因MYH7表达水平也极显著降低(P<0.01)。说明METTL21C促进小鼠成肌细胞分化,其通过调控MEF2的表达影响MRFS家族基因从而影响成肌分化。

To investigate the function of METTL21C in the differentiation of mouse myoblasts,shRNA interference sequences of two groups of METTL21C were designed,and the interference vector targeting METTL21C was constructed.C2C12 myoblasts were infected by lentivirus method,and C2C12 cell lines with stable interference with METTL21C were screened and interference efficiency was detected.qPCR and Western blot were used to detect the expression of genes related to myoblast differentiation after 2%horse serum was induced.The results showed that the two groups of lentivirus interference vectors targeting METTL21C were successfully constructed.qPCR and Western blot results showed that the expression level of METTL21C in the interference group was significantly decreased,and the interference efficiency of shRNA1 group was the highest(73.6%).In addition,the expression levels of MyoG,MyoD,Myf5,MRF4,the markers of myoblast differentiation,in the interference group were significantly decreased,and the expression level of MEF2C was more significantly down-regulated(P<0.01).The expression level of slow muscle marker MYH7 was also significantly reduced(P<0.01).The above results indicated that METTL21C promotes the differentiation of myoblasts in mice,and it affects the MRFS family genes by regulating the expression of MEF2,thus affecting the differentiation of myoblasts.