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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。
Objective To investigate the effect of targeting NUCB2 gene siRNA on the silencing of IAR20 NUCB2 gene in rat hepatocytes and the effect on insulin signaling pathway and apoptosis in rat hepatocytes. Methods siRNA targeting NUCB2 gene was constructed and transfected into IAR20 cells. The protein levels and phosphorylation of PEPCK, G-6-Pase, InsR, IRS-1 and Akt were detected by Western blot. The apoptosis of cells was detected by flow cytometry. The mRNA expression of p53 and caspase3 was detected by RT-PCR. Results After transfection with siRNA for 48 h, the expression of NUCB2 in IAR20 cells was significantly decreased (all P <0.05). PEPCK, G-6-Pase protein and mRNA expression was significantly increased, while IR, IRS-1, AKT phosphorylation decreased (P <0.01 or P <0.05). The apoptosis of IAR20 cells was significantly increased, the expression of p53 and caspase 3 mRNA and cleaved caspase 3 were significantly increased (P <0.01 or P <0.05). Conclusion NUCB2-specific siRNA can increase insulin resistance in rat hepatocytes by down-regulating IR / IRS-1 / Akt signaling pathway and increase apoptosis by up-regulating caspase 3 and p53 expression.