肿瘤坏死因子-α诱导蛋白3-受体作用蛋白-半胱氨酰天冬氨酸特异性蛋白酶-8凋亡传导在肝癌细胞对肿瘤坏死因子相关的凋亡诱导配体耐受中的作用

Tumor necrosis factor-α inducing protein 3-receptor-interacting protein-Caspase-8 complex in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis tolerance in hepatocellular carcinoma cells

目的 探讨肿瘤坏死因子-α诱导蛋白3（A20）及受体作用蛋白（RIP）沉默增加肝癌细胞HepG2对肿瘤坏死因子相关的凋亡诱导配体（TRAIL）敏感性的分子机制.方法 合成针对A20基因和RIP基因的特异性小干扰RNA（siRNA）并转染至肝癌细胞HepG2中,应用酸性磷酸酶（AP）法检测A20-siRNA或RIP-siRNA联合TRAIL对肝癌细胞HepG2生长的抑制作用以及Westernblot检测对半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-8蛋白表达的影响.结果 成功合成A20-siRNA和RIP-siRNA并转染入HepG2细胞中.A20-siRNA与TRAIL联合处理HepG2细胞可活化RIP、Caspase-8和Caspase-3,诱导细胞凋亡.RIP-siRNA联合TRAIL可以明显抑制HepG2细胞的增殖（P＜0.05）.另外RIP-siRNA联合TRAIL作用HepG2后可活化Caspase-8.结论 A20和RIP的表达是肝癌细胞对TRAIL耐受的主要原因,A20-siRNA或RIP-siRNA可以有效抑制肝癌HepG2细胞A20及RIP基因的表达,并增加TRAIL对肝癌HepG2细胞凋亡的诱导作用；其机制是通过“A20-RIP-Caspase-8”反应链序列调控活化下游Caspase-8,活化诱导的死亡受体传导通路来实现的.

Objective To investigate the molecular mechanism of silencing Tumor necrosis factorα inducing protein 3 （A20） and receptor-interacting protein （RIP） gene promoting tumor necrosis factorrelated apoptosis-inducing ligand （TRAIL）-induced apoptosis in hepatocellular carcinoma cell lines.Methods Synthesized A20-small interfering RNA （siRNA） and RIP-siRNA were transfected into HepG2 cells.The cell death of HepG2 was determined by acid phosphatase assay and the expression of Caspase-8 by Western blotting after A20-siRNA or RIP-siRNA combined with TRAIL treatment.Results A20-siRNA could activate RIP,Caspase-8,Caspase-3 and increase the sensitivity of HepG2 cells to TRAIL-induced apoptosis.The proliferation of HepG2 was inhibited by RIP-siRNA combined with TRAIL treatment （P 〈0.05）.Furthermore,Caspase-8 was activated after RIP-siRNA combined with TRAIL treatment.Conclusion A20-RIP-Caspase-8 complex could sensitize HepG2 to TRAIL-induced apoptosis through elimination of Caspase-8 inhibition.