NUCB2 siRNA对肝细胞胰岛素信号通路及细胞凋亡的影响

Effects of NUCB2 siRNA on insulin signaling pathway and cell apoptosis of hepatic cell

目的探讨靶向NUCB2基因特异性siRNA对大鼠肝细胞IAR20 NUCB2基因沉默效应、对大鼠肝细胞胰岛素信号通路及细胞凋亡的影响。方法构建合成靶向NUCB2基因的siRNA,转染IAR20细胞。Western blot检测PEPCK、G-6-Pase、InsR、IRS-1、Akt的蛋白含量及其磷酸化水平。采用流式细胞术检测细胞凋亡情况,使用RT-PCR检测p53及caspase3 mRNA水平。结果转染siRNA 48 h后,IAR20细胞NUCB2表达明显降低(P均<0.05)。PEPCK、G-6-Pase蛋白及mRNA表达量明显增加,同时IR、IRS-1、AKT的磷酸化表达降低(P<0.01或P<0.05)。IAR20细胞凋亡明显增加,p53及caspase 3 mRNA表达及cleaved caspase 3明显增加(P<0.01或P<0.05)。结论 NUCB2特异性siRNA能通过下调IR/IRS-1/Akt信号通路加重大鼠肝细胞胰岛素抵抗,并能够通过上调caspase 3及p53表达增加细胞凋亡。

To investigate the effects of NUCB2 siRNA on normal rat hepatic cell IAR20 and the effects of nesfatin-1 knockdown on insulin signaling pathway and cell apoptosis in IAR20 cell,NUCB2 siRNA was established and transfected in IAR20 cell.Then,protein expression levels of PEPCK,G-6-Pase,IR,P-IR,IRS-1,P-IRS-1,Akt,P-Akt were determined by Western blot;mRNA expression levels of PEPCK and G-6-Pase were determined by real-time quantitative PCR.Furthermore,cell apoptosis and mRNA levels of p53 and caspase 3 were also determined.We found that 48 hours after siRNA transfection,NUCB2 expression decreased significantly（P〈0.05）,while PEPCK and G-6-Pase mRNA and protein levels increased significantly in IAR20 cell（P〈0.01 or P〈0.05）.Meanwhile,phosphorylation levels of IR,IRS-1 and Akt decreased in IAR20 cell.In addition,NUCB2 siRNA also increased cell apoptosis,caspase 3 and p53 mRNA and cleaved caspase 3 expression levels（P〈0.01）.We came to the conclusion that NUCB2 siRNA increases insulin resistance through dampening the phosphorylation of InsR/IRS/Akt signaling pathway and increases cell apoptosis through increasing caspase 3 and p53 mRNA levels.