干扰素诱导蛋白16 siRNA对干扰素α诱导的人血管内皮细胞凋亡的影响

Effect of IFI16 siRNA on interferon-αinduced apoptosis in human vascular endothelial cells

目的:探讨干扰素诱导蛋白16(IFI16)siRNA对干扰素-α(IFN-α)诱导的人血管内皮细胞(HVECs)凋亡的影响及其机制。方法:应用转染IFN-α和(或)IFI16siRNA瞬时干预体外培养的HVECs,分别设空白组为阴性对照组,IFN-α加非特异性siRNA转染组为对照组,RT-PCR法检测IFI16mRNA表达,流式细胞仪Annexin-V FITC/PI法检测细胞凋亡,Western blotting检测蛋白表达及细胞增殖信号通路相关蛋白磷酸化水平。结果:与阴性对照组比较,对照组及IFN-α组IFI16mRNA和蛋白表达上调,细胞凋亡增多,伴RAS蛋白表达减少,RAF、ERK磷酸化水平下降(P<0.01);IFN-α加IFI16siRNA组IFI16mRNA及蛋白表达下调,细胞凋亡减少,RAS蛋白表达增多,RAF及ERK磷酸化水平升高(P<0.01)。与对照组比较,IFN-α加IFI16siRNA组IFI16mRNA和蛋白表达减少,细胞凋亡减少,伴Ras蛋白表达增多,RAF、ERK磷酸化水平上调(P<0.01);而IFN-α组上述指标差异无统计学意义。在上述过程中,P38及AKT蛋白磷酸化水平无明显变化。结论:RAS信号途径参与IFI16siRNA抑制IFN-α诱导的HVECs凋亡。

Objective：To study the inhibitory effects of IFI16 siRNA on IFN-α induced apoptosis in HVECs. Method：Cultured HVECs were treated with transfection IFN-α alone or together with IFI16 siRNA in vitro instan- taneously. The blank group was as negative control group,IFN-α and nonspecific siRNA transfection group was as control group. The expression of IFI16 mRNA was determined by semiquantitative RT-PCR. The cell apoptosis were analyzed by flow cytometry with Annexin-V FITC/PI method. The expression of IFI16, RAS protein and the phosphorylation levels of cell proliferation signaling pathway proteins were analyzed by Western blotting. Result.. Compared with negative control group, in control group and IFN-α induction group, the expression of IFI16 mR- NA and protein was up-regulated, the cell apoptosis was increased, the expression of RAS protein was decreased and the phosphorylation levels of RAF and ERK was dropped; In IFN-α induction and IFI16 siRNA transfaction group, the expression of IFI16 mRNA and protein was down-regulated, the cell apoptosis was decreased, the expression of RAS protein was increased and the phosphorylation levels of RAF and ERK was enhanced in IFN-α in- duction and IFI16 siRNA transfection group, Compared with control group, the expression of IFI16 mRNA and protein was decreased, the cell apoptosis was suppressed, the expression of RAS protein was increased and the phosphorylation levels of RAF and ERK was increased in IFN-α induction and IFI16 siRNA transfaction group. The difference of the targets of the above was no significance of statistics. In the process of the above, the phos- pborylation levels of P38 and AKT were no variation. Conclusion： RAS signaling pathway participation in IFI16 siRNA suppress IFN-α induced apoptosis in HVECs.