The Biological Function of Hepatitis B Virus X Protein in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the major malignant tumors that lead to death. Chronic hepatitis B virus infection is an important risk factor for HCC initiation. HBx protein, encoded by the HBV X gene, is a significant factor that promotes HBV-related HCC, although the exact molecular mechanism remains unclear. This article summarizes the pathological roles and related mechanisms of HBx in HCC. HBx plays a carcinogenic role by promoting cell proliferation, metastasis, and angiogenesis and inhibiting apoptosis in HCC. A detailed study of the biological functions of HBx will help to elucidate the mechanism of hepatocarcinogenesis and lead to the development of novel therapeutic targets for the treatment of HBV-related HCC.     

一种转导人原代肝细胞的LV-HBx诱导调控表达系统

目的：建立一种在人原代肝细胞中可诱导调控表达HBx的慢病毒表达系统,以便研究HBx生物学特性及其对人肝细胞功能的影响。方法：采用Tet-on系统构建四环素诱导调控的慢病毒-HBx表达系统,并用流式细胞术对其进行优化以达到较高的表达效率。应用该系统制备LV-HBx和LV-rtTA重组慢病毒共同转导肝癌细胞系HepG2和人原代肝细胞后,再加入强力霉素（Dox）诱导调控HBx表达,并采用实时荧光定量PCR（qPCR）检测人原代肝细胞中肿瘤转移相关基因1（MTA1）、血管内皮生长因子A（VEGFA）、转化生长因子β1（TGFB1）、上皮型钙黏附素1（CDH1）、胰岛素样生长因子结合蛋白3（IGFBP3）mRNA的表达水平。结果：重组慢病毒-HBx表达系统构建成功,LV-HBx和LV-rtTA重组慢病毒用量配比经过优化,采用1：1或2：1转导后可达到较高的表达效率,Dox诱导产生的表达HBx的阳性细胞率分别为67.3%和66.7%。应用该系统将HBx基因转导入肝癌细胞系HepG2和人原代肝细胞后,可用Dox进行诱导调控HBx的高效表达。其中人原代肝细胞中Dox诱导组的HBx表达水平约为对照组（不加Dox诱导）的45倍,经qPCR检测高表达HBx的人原代肝细胞中MTA1、VEGFA、TGFB1 mRNA的表达水平较对照组均显著升高（P均 < 0.05）,而CDH1、IGFBP3 mRNA表达水平显著降低（P均 < 0.05）。结论：成功建立了转导人原代肝细胞的LV-HBx四环素诱导调控表达系统,该系统的建立为研究HBx等乙肝病毒基因对人原代肝细胞的影响打下了基础。     

丹参多酚酸盐对HepG2215细胞X蛋白(HBx)表达的影响

目的:研究丹参多酚酸盐对HepG2215细胞X蛋白(HBx)表达的影响。方法:将HepG2215细胞分为对照组和药物组,药物组设三个浓度(100μg/ml2、00μg/ml4、00μg/ml)。待药物作用细胞24h后,荧光定量PCR法检测HepG2215细胞HBx mR-NA表达水平,ELISA法检测细胞内HBx蛋白的表达水平。结果:与对照组相比,药物组在不同的剂量100μg/ml2、00μg/ml、400μg/ml下均可明显降低HBx mRNA的表达水平,但不呈现剂量-反应关系。ELISA结果显示:药物组在不同的剂量100μg/ml2、00μg/ml、400μg/ml下均可明显降低胞内HBx蛋白表达的水平,且呈现剂量-反应关系。结论:丹参多酚酸盐可显著影响HepG2215细胞中X蛋白的表达。     

Could HBx Protein Expression Affect Signal Pathway Inhibition by Gefitinib or Selumetinib,a MEK Inhibitor,in Hepatocellular Carcinoma Cell Lines?

Hepatitis B virus X (HBx) protein has been known to play an important role in development of hepatocellular carcinoma (HCC). The aim of this study is to find out whether HBx protein expression affects antiproliferative effect of an epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor and a MEK inhibitor in HepG2 and Huh-7 cell lines. We established HepG2 and Huh-7 cells transfected stably with HBx gene. HBx protein expression increased pERK and pAkt expression as well as β-catenin activity in both cells. Gefitinib (EGFR-TK inhibitor) inhibited pERK and pAkt expression and β-catenin activity in both cells. Selumetinib (MEK inhibitor) reduced pERK level and β-catenin activity but pAkt expression was rather elevated by selumetinib in these cells. Reduction of pERK levels was much stronger with selumetinib than gefitinib in both cells. The antiproliferative efficacy of selumetinib was more potent than that of gefitinib. However, the antiproliferative effect of gefitinib, as well as selumetinib, was not different between cell lines with or without HBx expression. Signal pathway activation by HBx might not be strong enough to attenuate the antiproliferative effect of EGFR-TK inhibitor. Future experiments are needed to understand the role of HBx protein expression in HCC treatment using molecular targeting agent.     

稳定表达HBx的人肝星形细胞系的建立及其生物学特性初探

目的建立稳定表达HBVX蛋白（HBx）的LX-2细胞系,并初步研究HBx对于肝星形细胞（HSC）的增殖及其I型胶原蛋白表达的影响。方法构建包含HBx基因的重组慢病毒载体LV5-X,并转染LX-2细胞,同时用对照质粒转染LX-2细胞,经嘌呤霉素筛选后获得HBx稳定表达的LX-2-X和对照LX-2-C细胞系。荧光显微镜下观察感染效率,Western印迹检测细胞株中HBx的表达。在LX-2细胞系的生物学特性探索方面,应用CCK_8法检测细胞的增殖;Real—timePCR检测细胞中I型胶原蛋白mRNA的表达;ELISA法愉测细胞培养上清中I型胶原蛋白的表达。结果成功构建重组慢病载体LV5-X,感染LX-2细胞后建立细胞系LX-2-X和LX-2-C,嘌呤霉素筛选3d后,荧光显微镜显示近100％细胞发出绿色荧光,Western印迹证实LX-2-X细胞稳定表达HBx。培养72h和96h后LX-2-C细胞增殖明显高于LX-2和LX-2-C细胞组（P〈0．05）;PCR证实LX-2X细胞I型胶原蛋白mRNA合成高于LX-2和LX-2-C细胞组（P〈0．05）;ELISA结果显示LX一2一X细胞I型胶原蛋白表达高于LX-2和LX-2-C细胞组（P〈0．05）。结论成功构建了稳定表达HBx的LX-2细胞系;HBx能够促进HSC的增殖,并增加I型胶原蛋白的表达。     

Effects of Rubiadin isolated from Prismatomeris connata on anti‐hepatitis B virus activity in vitro

Prismatomeris connata was a kind of Rubiaceae plant for treatment of hepatitis, hepatic fibrosis and silicosis. Whereas, the effective components of Prismatomeris connata remains unexplored. The aim of this study was to investigate the inhibitory effects and mechanisms of Rubiadin isolated from Prismatomeris connata against HBV using HepG2.2.15 cells. The levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme‐linked immunosorbent assay. HBV DNA was qualified q‐PCR. Rubiadin was isolated by silica gel column. The structure of the compound was elucidated by HPLC, FT‐IR, ¹H‐NMR, ¹³C‐NMR and identified as 1,3‐Dihydroxy‐2‐methyl‐9, 10‐anthraquinone. Rubiadin significantly decreased HBeAg,HBcAg secretion level and inhibit HBV DNA replication. Rubiadin inhibits the proliferation of the cells and HBx protein expression in a dose‐dependent manner. The intracellular calcium concentration was significantly reduced. These results demonstrated that Rubiadin could inhibit HepG2.2.15 cells proliferation, reduce the level of HBx expression, and intracellular free calcium, which might become a novel anti‐HBV drug candidate.     

HBx蛋白抑制DKK4表达的机制及对肝癌细胞系增殖和迁移的影响

目的 探讨乙型肝炎病毒X蛋白下调DKK4的机制及对肝癌细胞系增殖和迁移能力的影响。方法 将编码乙型肝炎病毒X蛋白的腺病毒（Ad-HBx）分别感染肝癌细胞系HepG2和SMMC7721细胞,同时设感染GFP腺病毒（Ad-GFP）为对照组,用去乙酰化酶抑制剂（TSA）处理肝癌细胞系,并用小干扰RNA-组蛋白去乙酰化酶1（si-HDAC1）及过表达DKK4的慢病毒转染肝癌细胞系。Western blot检测DKK4、HDAC1及SIRT1的表达情况。MTT实验、结晶紫实验和Transwell实验分别检测肝癌细胞系的增殖和迁移能力。结果 Ad-HBx感染肝癌细胞系后DKK4蛋白表达水平下降（P<0.05）,TSA处理后DKK4蛋白表达水平回复（P<0.05）。Ad-HBx感染肝癌细胞系后,HDAC1及SIRT1蛋白水平显著升高（P<0.05）。小干扰RNA沉默HDAC1后能够恢复DKK4的表达（P<0.05）,沉默HDAC1和（或）过表达DKK均能抑制肝癌细胞系的增殖和迁移能力（P<0.05）。结论 乙型肝炎病毒X蛋白通过上调HDAC1、SIRT1来抑制DKK4的表达,沉默HDAC1及过表达DKK4蛋白表达能够抑制感染Ad-HBx肝癌细胞系的增殖和迁移能力。