TNF-α通过内质网应激信号通路诱导肝癌细胞自噬并促进增殖的研究

摘要

Objective To investigate the effect of tumor necrosis factor-α (TNF-α) upon the autophagy and proliferation of liver cancer cells through endoplasmic reticulum stress signaling pathway.Methods The expression levels of TNF-oα,BECN1,LC3B and PCNA in the cancerous and paracancerous tissues of patients with primary hepatocellular carcinoma,and the normal tissues adjacent to tumors of patients with hepatic hemangioma were determined by immunohistochemistry.After the HepG2 was treated with recombinant human TNF-α cytokine,the expression of autophagy-related proteins of BECN1 and LC3B,and the cell proliferation marker of PCNA were quantitatively measured by Western blot.After the treatment with autophagy inhibitor 3-MA,the changes in the HepG2 cell viability and PCNA expression were observed following TNF-α stimulation.After the HepG2 cells were treated with TNF-α,the expression levels of eIF2a and p-eIF2a related to the ER stress signaling pathway were detected by Western blot.Results The expression levels of TNF-α,BECN1,LC3B and PCNA proteins in the cancerous and paracancerous tissues of hepatocellular carcinoma patients were significantly up-regulated compared with those in the normal liver tissue.Following the HepG2 cells were treated with TNF-α,the expression levels of BECN1,LC3B and PCNA proteins were up-regulated,and the cell viability was increased.After the treatment with autophagy inhibitor 3-MA,the expression of PCNA protein and cell viability were down-regulated.After the treatment with TNF-α,the phosphorylated level of eIF2a was significantly enhanced.Conclusion TNF-α can induce the autophagy of liver cancer cells via the endoplasmic reticulum stress signaling pathway,thereby promoting the proliferation of liver cancer cells.%目的 研究肿瘤坏死因子-α(TNF-α)通过内质网应激信号通路对肝癌细胞自噬和增殖的作用.方法 利用免疫组织化学检测原发性肝细胞癌患者癌灶组织、配对的癌旁组织,以及肝血管瘤患者瘤旁正常肝组织中TNF-α、BECN1、LC3B、PCNA的表达水平.用人源TNF-α重组因子刺激肝癌细胞株HepG2后,通过蛋白免疫印迹法检测自噬相关蛋白BECN1、LC3B以及细胞增殖标志物PCNA表达水平,并利用自噬抑制剂3-MA抑制HepG2细胞自噬后,检测TNF-α刺激后的细胞活性及PCNA蛋白水平的改变情况.通过蛋白免疫印迹法检测TNF-α刺激HepG2细胞后内质网应激通路相关蛋白eIF2α、p-eIF2α的表达水平.结果 肝细胞癌患者癌灶和癌旁组织中TNF-α、BECNI、LC3B、PCNA蛋白表达量均较正常人肝组织升高.TNF-α刺激HepG2细胞后,细胞内BECN1、LC3B、PCNA蛋白表达水平升高,细胞活性增强,使用3-MA抑制HepG2细胞自噬后,细胞PCNA蛋白表达水平以及细胞活性下降,TNF-α刺激HepG2细胞后内质网应激活性标志物eIF2αα磷酸化水平明显增加.结论 TNF-α通过内质网应激信号通路诱导肝癌细胞自噬,从而促进肝癌细胞的增殖.