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| 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 |
| 况钦,吴勇,杜彬,毋楠,周梦瑶,杨鑫,卢杨,冯涛* |
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(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
*通信作者) |
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| 摘要: |
| 目的 探讨miRNA-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qPCR检测miRNA-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miRNA-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miRNA-544-3p对HBx-EGFP-14-19细胞迁移和侵袭的影响，采用qPCR和蛋白质印迹法探究miRNA-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（mTOR）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miRNA-544-3p表达水平均降低（P均<0.05）。与对照组比较，转染miRNA-544-3p mimic的HBx-EGFP-14-19细胞迁移和侵袭能力均降低，转染miRNA-544-3p inhibitor的HBxEGFP-14-19细胞迁移和侵袭能力均增强（P均<0.05）。qPCR和蛋白质印迹法检测结果显示miRNA-544-3p低表达与Arf6表达水平上调、Akt-mTOR信号轴的激活有关。结论 HBx可能通过下调miRNA-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。 |
| 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 |
| DOI：10.16781/j.CN31-2187/R.20211034 |
| 投稿时间：2021-10-15修订日期：2022-02-24 |
| 基金项目:国家自然科学基金面上项目（81071770）. |
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| Hepatitis B virus X protein downregulates microRNA-544-3p and activates the Arf6/Akt-mTOR signaling axis to promote liver cell migration and invasion |
| 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
*Corresponding author) |
| Abstract: |
| Objective To investigate the effect of microRNA (miRNA)-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 (qPCR) was used to detect the expression of miRNA-544-3p in HBx-EGFP-14-19 cells and liver tissues of mouse models. miRNA-544-3p mimics and inhibitors were transiently transfected into HBx-EGFP-14-19 cells. The effects of miRNA-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. qPCR and Western blotting were used to explore the regulatory effect of miRNA-544-3p on the expression of adenosine diphosphate ribosylation factor 6 (Arf6), protein kinase B (Akt) and mammalian target of rapamycin (mTOR). 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 miRNA-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 miRNA-544-3p mimics were decreased, and the migration and invasion abilities of HBx-EGFP-14-19 cells transfected with miRNA-544-3p inhibitors were increased (all P<0.05). The results of qPCR and Western blotting showed that the low expression of miRNA-544-3p was correlated with the up-regulation of Arf6 expression and the activation of Akt-mTOR signal axis. Conclusion HBx may promote the invasion and migration of mouse hepatocarcinoma cells by downregulating miRNA-544-3p and activating Arf6/Akt-mTOR signal axis. |
| Key words: hepatitis B virus X protein hepatocellular carcinoma microRNA-544-3p adenosine diphosphate ribosylation factor 6 protein kinase B mammalian target of rapamycin |
