HBx对HBV相关性肝癌增殖和迁移能力的影响及其机制

doi:10.3877/cma.j.issn.2095-3232.2022.02.017

中华肝脏外科手术学电子杂志 ›› 2022, Vol. 11 ›› Issue (02) : 198 -202. doi: 10.3877/cma.j.issn.2095-3232.2022.02.017

基础研究

HBx对HBV相关性肝癌增殖和迁移能力的影响及其机制

汤永昌¹, 袁峰¹, 梁豪², 钟昭众¹, 熊志勇², 曹明波¹, 任昱朋¹, 李宇轩¹, 姚志成², 邓美海¹^,^†(

)

^1. 510630　广州，中山大学附属第三医院肝胆外科
^2. 510530　广州，中山大学附属第三医院岭南医院普通外科

收稿日期:2021-12-22 出版日期:2022-04-10
通信作者: 邓美海
基金资助:
广东省自然科学基金(2016A030313848); 广州市科技计划项目(201704020175)

Effect of HBx on proliferation and migration of HBV-associated hepatocellular carcinoma and its mechanism

Yongchang Tang¹, Feng Yuan¹, Hao Liang², Zhaozhong Zhong¹, Zhiyong Xiong², Mingbo Cao¹, Yupeng Ren¹, Yuxuan Li¹, Zhicheng Yao², Meihai Deng¹^,^†()

^1. Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
^2. Department of General Surgery, Lingnan Hospital, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510530, China

Received:2021-12-22 Published:2022-04-10
Corresponding author: Meihai Deng

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引用本文：

汤永昌, 袁峰, 梁豪, 钟昭众, 熊志勇, 曹明波, 任昱朋, 李宇轩, 姚志成, 邓美海. HBx对HBV相关性肝癌增殖和迁移能力的影响及其机制[J/OL]. 中华肝脏外科手术学电子杂志, 2022, 11(02): 198-202.

Yongchang Tang, Feng Yuan, Hao Liang, Zhaozhong Zhong, Zhiyong Xiong, Mingbo Cao, Yupeng Ren, Yuxuan Li, Zhicheng Yao, Meihai Deng. Effect of HBx on proliferation and migration of HBV-associated hepatocellular carcinoma and its mechanism[J/OL]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2022, 11(02): 198-202.

目的

探讨乙型肝炎病毒X蛋白（HBx）对HBV相关性肝细胞癌（肝癌）增殖和迁移能力的影响及其作用机制。

方法

构建稳定过表达HBx的肝癌细胞系（SMMC7721、Huh7），采用CCK-8法、细胞平板克隆实验和Transwell迁移实验检测HBx在肝癌细胞增殖和迁移能力中的作用。microRNA（miRNA，miR）芯片数据分析HBV相关性肝癌差异表达的miRNA。荧光定量PCR检测lncRNA PTENpg1、miR-21的表达情况，Western blot法检测PTEN的表达情况。构建过表达miR-21的肝癌细胞系（SMMC7721、Huh7），采用CCK-8法、细胞平板克隆实验和Transwell迁移实验检测miR-21对肝癌细胞增殖和迁移能力的影响。

结果

细胞平板克隆形成实验、CCK-8法检测和Transwell迁移实验显示，过表达HBx促进肝癌细胞克隆形成、生长和迁移。miRNA芯片数据分析显示miR-21为HBV相关性肝癌的差异表达基因。荧光定量PCR检测显示，HBx过表达后，肝癌细胞lncRNA PTENpg1表达明显下调，miR-21表达明显上调。Western blot检测显示，HBx和miR-21过表达后PTEN蛋白水平下调。细胞平板克隆形成实验、CCK-8法检测和Transwell迁移实验显示，上调miR-21促进肝癌细胞的增殖、迁移能力。

结论

HBx可能通过调控lncRNA PTENpg1-miR-21-PTEN分子信号通路，促进HBV相关性肝癌发生、发展。

关键词: 癌，肝细胞, 肝炎病毒，乙型, 乙型肝炎病毒X蛋白, 微RNAs, 细胞增殖, 细胞迁移

Objective

To evaluate the effect of hepatitis B virus X protein (HBx) on the proliferation and migration of HBV-associated hepatocellular carcinoma (HCC) and the mechanism.

Methods

The HCC cell lines (SMMC7721 and Huh7) stably overexpressing HBx were constructed. The effect of HBx upon the proliferation and migration of HCC cells was assessed by CCK-8 assay, plate cloning experiment and Transwell chamber test. The differentially-expressed microRNAs (miRNAs, miR) inHBV-associated HCC were identified by miRNA chip data. The expression levels of lncRNA PTENpg1 and miR-21 were measured by fluorescent quantitative PCR. The expression level of PTEN was detected by Western blot. The HCC cell lines (SMMC7721 and Huh7) overexpressing miR-21 were constructed. The effect of miR-21 on the proliferation and migration of HCC cells was evaluated by CCK-8 assay, plate cloning experiment and Transwell chamber test.

Results

Plate cloning experiment, CCK-8 assay and Transwell chamber test showed that overexpression of HBx promoted the colony formation, proliferation and migration of HCC cells. Analysis of siRNA chip data demonstrated that miR-21 was a differentially-expressed gene of HBV-associated HCC. Fluorescent quantitative PCR revealed that the expression level of lncRNA PTENpg1 was significantly down-regulated, whereas that of miR-21 was significantly up-regulated after HBx overexpression. Western blot showed that the expression level of PTEN protein was down-regulated while the HBx and miR-21 were over expressed. Plate cloning experiment, CCK-8 assay and Transwell chamber test indicated that up-regulation of miR-21 promoted the proliferation and migration of HCC cells.

Conclusions

HBx can promote the incidence and progression of HBV-associated HCC by regulating the lncRNA PTENpg1-miR-21-PTEN molecular signaling pathway.

Key words: Carcinoma, hepatocellular, Hepatitis B virus, Hepatitis B virus X protein, MicroRNAs, Cell proliferation, Cell migration

图1 HBx对肝癌细胞增殖、迁移能力的影响注a、b、c分别为细胞平板克隆形成实验、CCK-8法检测、Transwell迁移实验，示过表达HBx促进肝癌细胞克隆形成、细胞生长和迁移；*为P<0.05；Vector、HBx分别为空载体对照组和HBx过表达组；HBx为乙型肝炎病毒X蛋白

图2 HBx对lncRNA PTENpg1-miR-21-PTEN信号通路的影响注a、b为荧光定量PCR检测显示肝癌细胞过表达HBx，lncRNA PTENpg1表达下调，miR-21表达上调；c为Western blot检测显示肝癌细胞过表达HBx和miR-21，PTEN表达均下调；*为P<0.05；Control、Vector、HBx、miR-21分别为阴性对照组、空载体对照组、HBx过表达组、miR-21过表达组；HBx为乙型肝炎病毒X蛋白

图3 miR-21对肝癌细胞增殖和迁移能力的影响注a、b、c分别为细胞平板克隆形成实验、CCK-8法检测、Transwell迁移实验，示miR-21过表达促进肝癌细胞克隆形成、细胞生长和迁移；*为P<0.05；Vector、miR-21分别为空载体对照组和miR-21过表达组

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