乙型肝炎病毒x蛋白通过作用MDM2对肝癌干细胞特性的调控及机制研究

Liver cancer stem cells play an important role in the development of hepatocellular carcinoma (HCC). HBx protein encoded by Hepatitis B virus (HBV） is closely related to HCC. However, whether HBx can regulate stem-like characteristics of liver cancer stem cells and its molecular mechanism is unknown. In previous studies, we first proved that HBx can induce malignant transformation and tumorigenicity of hepatic progenitor cells, and HBx expression was positively correlated with markers of liver cancer stem cells in HCC specimens(Hepatology 2012). Recently, we found that hepatoma cell lines with over-expression of HBx showed more obvious stem-like characteristics. Furthermore, gene expression profile microarray analysis showed that MDM2, a key regulator in p53 degradation, was significantly up-regulated in suspension cultured spheres of hepatoma cells with over-expression of HBx, which suggested that MDM2 was a critical downstream target gene of HBx in promoting stem-like characteristics of liver cancer. It is well-known that p53 can inhibit self-renewal ability of stem cell, and p53 mutation have been detected only in 50% of liver cancer, which suggests that HBV may inhibit expression and function of p53 via HBx-MDM2 pathway. This study will in-depth study the regulation relationship among HBx-MDM2-p53 and examine the role of HBx-MDM2-p53 in maintaining stem-like properties of liver cancer stem cells, which will further elucidates significance and molecular mechanisms of liver cancer stem cells in the development of HBV-related liver cancer.?In addition, our study will investigate clinical significance of HBx/MDM2 pathway in diagnosis and prognosis of patients with HCC.

肝癌干细胞在肝癌的发生和发展中发挥着重要作用，由HBV编码的HBx蛋白与肝癌关系密切,但HBx是否能调控肝癌干细胞特性及其分子机制尚待研究。前期我们首次证明HBx可促进肝前体细胞恶性转化诱发肝癌，在肝癌标本中HBx与肝癌干细胞标志物呈正相关（Hepatology 2012）。最近我们发现过表达HBx的肝癌细胞系表现出更明显的干细胞特性，通过基因表达谱芯片分析进一步发现引起p53降解的关键调控分子MDM2在过表达HBx的肝癌细胞悬浮肿瘤球中显著上调，已知p53可抑制干细胞特性，且仅在50%肝癌中检出突变，结合本发现提示HBV可能通过HBx对MDM2的诱导来抑制p53表达及功能。本课题拟深入研究HBx-MDM2-p53之间的调控关系及其对肝癌干细胞特性的调控作用，以进一步阐明肝癌干细胞在HBV相关肝癌的发生和发展中的意义及分子机制，并探讨在HBx/MDM2通路在肝癌诊断和预后中的临床意义。

肝癌的发病率及病死率较高，即使经手术治疗，肝癌仍然容易复发和转移，因此，肝癌严重地威胁着人类的健康。乙型肝炎病毒（HBV）是肝癌的主要病因之一，我国的肝癌大部分由HBV感染所致，被称为HBV相关肝癌。大量研究表明，HBV与肝癌的发生及发展密切相关，因此阐明HBV相关分子机制对改善肝癌患者的预后至关重要。而我们的前期研究表明：1）HBV编码的HBx蛋白可诱导肝前体细胞恶性转化从而诱导肝癌的发生（Hepatology，2012）；2）OV6阳性的肝癌细胞具有肿瘤干细胞特性，并且可促进肝癌的侵袭和转移（Journal of Hepatology，2012）。本项目是在前期研究的基础上，进一步阐明HBV编码的HBx蛋白对OV6阳性肝癌干细胞特性的调控及机制研究，并明确HBx及肝癌干细胞标志物在肝癌患者预后评估中的临床意义。我们首先通过HBV相关肝癌临床标本验证了HBx与OV6表达水平存在正相关性，同时二者的联合表达与肝癌患者的临床病理特点如肿瘤大小、数目、微血管侵犯等明确相关，并且与肝癌患者的复发和5年生存率明确相关。其次，我们证明了HBx可促进OV6阳性肝癌干细胞的特性如自我更新、成瘤性、耐药性等，并且通过基因表达谱芯片及生物信息学分析，我们发现HBx是通过癌基因MDM2且不依赖于p53的通路来调控OV6阳性肝癌干细胞的特性。接着，我们深入地研究了相关分子机制：HBx可与MDM2直接相互作用并抑制MDM2的自身泛素化降解，促进MDM2表达及入核增加，增强CXCL12/CXCR4的转录，从而激活Wnt/β-catenin通路，最终促进了OV6阳性肝癌干细胞的特性。最后，本项目阐明了HBx/MDM2/CXCR4/OV6在HBV相关肝癌患者的预后评估中具有重要的临床价值。本项目为阐明HBV相关肝癌的发生及发展机制提供了新的理论依据，并为HBV相关肝癌患者的预后评估提供了新的指标。