乙型肝炎病毒X蛋白下调微RNA-544-3p并激活Arf6/Akt-mTOR信号轴促进肝脏细胞迁移和侵袭  

Hepatitis B virus X protein downregulates microRNA-544-3p and activates the Arf6/Akt-mTOR signaling axis to promote liver cell migration and invasion

作　　者：况钦 吴勇 杜彬 毋楠 周梦瑶 杨鑫[1] 卢杨 冯涛[1] KUANG Qin;WU Yong;DU Bin;WU Nan;ZHOU Meng-yao;YANG Xin-yue;LU Yang;FENG Tao(School of Pharmacy,Chongqing Medical University,Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学药学院、重庆市生化与分子药理学重点实验室,重庆400016

出　　处：《海军军医大学学报》2022年第6期604-613,共10页Academic Journal of Naval Medical University

基　　金：国家自然科学基金面上项目(81071770)。

摘　　要：目的探讨mi RNA-544-3p对乙型肝炎病毒X蛋白(HBx)诱导的肝细胞癌的影响及分子机制。方法培养含增强型绿色荧光蛋白(EGFP)和HBx的慢病毒稳转小鼠永生化肝前体细胞株14-19(HBx-EGFP-14-19细胞),并通过向昆明小鼠肝门静脉注射上述细胞构建可长期稳定表达HBx的模型,采用q PCR检测mi RNA-544-3p在HBxEGFP-14-19细胞及小鼠模型肝组织中的表达。在HBx-EGFP-14-19细胞中瞬时转染mi RNA-544-3p模拟物(mimic)或抑制剂(inhibitor),应用划痕愈合实验、Transwell细胞迁移和侵袭实验检测mi RNA-544-3p对HBx-EGFP-14-19细胞迁移和侵袭的影响,采用q PCR和蛋白质印迹法探究mi RNA-544-3p对二磷酸腺苷核糖基化因子6(Arf6)、Akt、哺乳动物雷帕霉素靶蛋白(m TOR)表达的调控作用。结果成功构建了可长期稳定表达HBx的小鼠模型,该小鼠在造模后360 d处死时可见肝组织有恶性肿瘤生成。与对照组相比,HBx-EGFP-14-19细胞和HBx模型小鼠各时间点(造模后30、90、180、360 d)肝组织中mi RNA-544-3p表达水平均降低(P均<0.05)。与对照组比较,转染mi RNA-544-3p mimic的HBx-EGFP-14-19细胞迁移和侵袭能力均降低,转染mi RNA-544-3p inhibitor的HBxEGFP-14-19细胞迁移和侵袭能力均增强(P均<0.05)。q PCR和蛋白质印迹法检测结果显示mi RNA-544-3p低表达与Arf6表达水平上调、Akt-m TOR信号轴的激活有关。结论HBx可能通过下调mi RNA-544-3p激活Arf6/AktmTOR信号轴促进小鼠肝癌细胞的侵袭和迁移能力。Objective To investigate the effect of micro RNA(mi RNA)-544-3p on hepatitis B virus X protein(HBx)induced hepatocellular carcinoma and its molecular mechanism.Methods By injecting HBx-enhanced green fluorescent protein(EGFP)-14-19 cells into the hepatic portal vein of Kunming mice,an animal model with long-term stable expression of HBx was constructed.Quantitative polymerase chain reaction(q PCR)was used to detect the expression of mi RNA-544-3p in HBx-EGFP-14-19 cells and liver tissues of mouse models.mi RNA-544-3p mimics and inhibitors were transiently transfected into HBx-EGFP-14-19 cells.The effects of mi RNA-544-3p on the migration and invasion of HBx-EGFP-14-19 cells were detected by scratch wound healing assay and Transwell cell migration and invasion assay.q PCR and Western blotting were used to explore the regulatory effect of mi RNA-544-3p on the expression of adenosine diphosphate ribosylation factor 6(Arf6),protein kinase B(Akt)and mammalian target of rapamycin(m TOR).Results A mouse model that can stably express HBx for a long time was successfully constructed,and malignant tumors could be found in the liver tissues when the mice were sacrificed 360 d after modeling.Compared with the control group,the expression levels of mi RNA-544-3p in HBx-EGFP-14-19 cells and HBx model mice at different time points(30,90,180,and 360 d after modeling)were decreased(all P<0.05).Compared with the control group,the migration and invasion abilities of HBx-EGFP-14-19 cells transfected with mi RNA-544-3p mimics were decreased,and the migration and invasion abilities of HBx-EGFP-14-19 cells transfected with mi RNA-544-3p inhibitors were increased(all P<0.05).The results of q PCR and Western blotting showed that the low expression of mi RNA-544-3p was correlated with the up-regulation of Arf6 expression and the activation of Akt-m TOR signal axis.Conclusion HBx may promote the invasion and migration of mouse hepatocarcinoma cells by downregulating mi RNA-544-3p and activating Arf6/Akt-m TOR signal axis.

关 键 词：乙型肝炎病毒X蛋白 肝细胞癌 微RNA-544-3p 二磷酸腺苷核糖基化因子6 蛋白激酶B 哺乳动物雷帕霉素靶蛋白 

分 类 号：R735.7[医药卫生—肿瘤]