Ide基因通过AKT调控成肌细胞增殖和分化的研究

The Study of Ide Gene Regulating Myoblast Proliferation and Differentiation through AKT

旨在了解胰岛素降解酶(insulin-degrading enzyme,Ide)在猪不同组织中的表达情况,及其在C2C12成肌细胞增殖和分化中的作用。本研究利用RT-PCR和Western blotting技术检测了Ide在8月龄雄性小型猪不同组织中的表达;利用siRNA技术干扰Ide表达,检测了Ide在C2C12成肌细胞增殖、凋亡和分化中的作用,试验分为Ide-siRNA处理组和NC-siRNA(negative control)对照组,每组3个重复。结果显示,Ide在猪的不同组织(包括大脑、股四头肌、股二头肌、背最长肌、心、肝、脾、肺、肾和睾丸)中广泛表达,其中,在股四头肌、肾和睾丸中表达量相对更高。Ide-siRNA转染成肌细胞48 h后,Ide表达量极显著下降(P<0.001)。CCK-8检测显示,干扰Ide表达促进了成肌细胞的增殖,细胞周期相关基因表达也显著升高,同时发现干扰Ide表达未引起成肌细胞凋亡。利用2%马血清诱导成肌细胞分化的同时转染Ide-siRNA以干扰其表达,在分化的第2和5天发现,与对照组相比,分化相关基因Myog、Myhc等在干扰组中的表达均显著下降,提示成肌细胞的分化受到了抑制。进一步的研究还发现,在分化的第5天,Akt 2和磷酸化的AKT(P-AKT)表达量下降。综上,Ide在猪的不同组织中广泛表达,以肌肉、肾和睾丸中表达量更高。干扰Ide表达促进C2C12成肌细胞的增殖,分化时干扰Ide表达则通过Akt 2/P-Akt/Myog途径抑制C2C12成肌细胞的分化。

This study was performed to investigate the expression of insulin-degrading enzyme(Ide)in different tissues of pig as well as its role in C2C12 myoblast proliferation and differentiation.RT-PCR and Western blotting were used to detect the expression of Ide in different tissues of 8-month old male mini-pigs.siRNA technology was used to knock down the expression of Ide in mouse C2C12 cell line to explore the role of Ide in myoblast proliferation,apoptosis and differentiation.Two groups containing Ide-siRNA treatment group and NC-siRNA(negative control)group were set up.There were 3 replicates in each group.The results showed that Ide was widely expressed in brain,quadriceps,biceps femoris,longest dorsal muscle,heart,liver,spleen,lung,kidney,and testis of pigs,among which its expression was relatively higher in quadriceps,kidney,and testis.After Ide-siRNA transfection into myoblasts for 48 hours,Ide expression was extremely significantly decreased(P<0.001).CCK-8 analysis showed that myoblast proliferation was promoted after Ide interference.Accordingly,the expressions of cell cycle-related genes were significantly increased.Meanwhile,the down-regulation of Ide by siRNA interference did not lead to myoblast apoptosis.Myoblasts induced to differentiation by 2%horse serum were transfected with Ide-siRNA,which resulted in the significant decrease in expression of differentiation marker genes Myog and Myhc at Day 2 and Day 5 compared with control group.This implied that the differentiation of myoblasts was inhibited after Ide interference.We further found that the expressions of Akt 2 and phosphorylated AKT(P-AKT)were decreased at Day 5 of differentiation.In summary,Ide was widely expressed in different tissues of pigs,with higher expression in muscle,kidney and testis.Ide is involved in negatively regulating the proliferation of myoblasts,and interfering Ide can inhibit the differentiation of myoblasts through Akt 2/P-Akt/Myog pathway.