Zhang Weiying, Lu Zhanping, Kong Guangyao, Gao Yuen, Wang Tao, Wang Qi, Cai Na, Wang Honghui, Liu Fabao, Ye Lihong, Zhang Xiaodong
State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P,R, China.
Mol Cancer. 2014 May 28;13:128. doi: 10.1186/1476-4598-13-128.
BACKGROUND: Hepatitis B virus X protein (HBx) plays crucial roles in hepatocarcinogenesis. However, the underlying mechanism remains elusive. We have reported that HBx is able to up-regulate survivin in hepatocellular carcinoma tissues. The oncopreotein hepatitis B X-interacting protein (HBXIP), a target of miR-520b, is involved in the development of cancer. In this study, we focus on the investigation of hepatocarcinogenesis mediated by HBx. METHODS: The expression of HBx and survivin was examined in the liver tissues of HBx-Tg mice. The effect of HBx/survivin on the growth of LO2-X-S cells was determined by colony formation and transplantation in nude mice. The effect of HBx/survivin on promoter of miR-520b was determined by Western blot analysis, luciferase reporter gene assay, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP), respectively. The expression of HBx, survivin and HBXIP was detected by immunohistochemistry and real-time PCR in clinical HCC tissues, respectively. The DNA demethylation of HBXIP promoter was examined. The functional influence of miR-520b and HBXIP on proliferation of hepatoma cells was analyzed by MTT, colony formation, EdU and transplantation in nude mice in vitro and in vivo. RESULTS: In this study, we provided evidence that HBx up-regulated survivin in the liver cancer tissues of HBx-Tg mice aged 18 M. The engineered LO2 cell lines with survivin and/or HBx were successfully established, termed LO2-X-S. MiR-520b was down-regulated in LO2-X-S cells and clinical HCC tissues. Our data revealed that HBx survivin-dependently down-regulated miR-520b through interacting with Sp1 in the cells. HBXIP was highly expressed in LO2-X-S cells, liver cancer tissues of HBx-Tg mice aged 18 M and clinical HCC tissues (75.17%, 112/149). The expression level of HBXIP was positively associated with those of HBx or survivin in clinical HCC tissues. In addition, we showed that HBx survivin-dependently up-regulated HBXIP through inducing demethylation of HBXIP promoter in LO2-X-S cells and clinical HCC tissues. In function, low level miR-520b and high level HBXIP mediated by HBx with partner survivin contributed to the growth of LO2-X-S cells in vitro and in vivo. CONCLUSION: HBx accelerates hepatocarcinogenesis with partner survivin through modulating tumor suppressor miR-520b and oncoprotein HBXIP.
背景：乙型肝炎病毒X蛋白（HBx）在肝癌发生过程中发挥着关键作用。然而，其潜在机制仍不清楚。我们曾报道HBx能够上调肝癌组织中的生存素。癌蛋白乙型肝炎X相互作用蛋白（HBXIP）是miR-520b的一个靶点，参与癌症的发展。在本研究中，我们着重调查由HBx介导的肝癌发生。 方法：检测HBx-Tg小鼠肝脏组织中HBx和生存素的表达。通过集落形成和裸鼠移植来确定HBx/生存素对LO2-X-S细胞生长的影响。分别通过蛋白质免疫印迹分析、荧光素酶报告基因检测、免疫共沉淀（co-IP）和染色质免疫沉淀（ChIP）来确定HBx/生存素对miR-520b启动子的影响。分别通过免疫组织化学和实时定量PCR检测临床肝癌组织中HBx、生存素和HBXIP的表达。检测HBXIP启动子的DNA去甲基化情况。通过MTT、集落形成、EdU以及体外和体内裸鼠移植实验分析miR-520b和HBXIP对肝癌细胞增殖的功能影响。 结果：在本研究中，我们提供证据表明，在18月龄的HBx-Tg小鼠肝癌组织中HBx上调了生存素。成功构建了带有生存素和/或HBx的工程化LO2细胞系，命名为LO2-X-S。在LO2-X-S细胞和临床肝癌组织中miR-520b表达下调。我们的数据显示，在细胞中HBx通过与Sp1相互作用以生存素依赖的方式下调miR-520b。HBXIP在LO2-X-S细胞、18月龄的HBx-Tg小鼠肝癌组织以及临床肝癌组织（75.17%，112/149）中高表达。在临床肝癌组织中，HBXIP的表达水平与HBx或生存素的表达水平呈正相关。此外，我们表明在LO2-X-S细胞和临床肝癌组织中，HBx通过诱导HBXIP启动子去甲基化以生存素依赖的方式上调HBXIP。在功能上，由HBx与生存素共同介导的低水平miR-520b和高水平HBXIP促进了LO2-X-S细胞在体外和体内的生长。 结论：HBx与生存素共同作用，通过调节肿瘤抑制因子miR-520b和癌蛋白HBXIP来加速肝癌发生。
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