乙型肝炎病毒转录调控TIM-3促进肝癌增殖及免疫逃逸的分子机制研究

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer mortality. 80% of HCCs are linked to chronic infections with the hepatitis B virus (HBV) or hepatitis C virus. China is one of the HCC high-incidence districts which takes up 55% of HCC cases in the world. Chronic hepatitis B virus infection has been identified as a leading risk factor for HCC in China. So it is definitely urgent to explore the accurate mechanisms of HBV associated liver diseases..T cell immunoglobulin and mucin-domain-containing molecule 3 (TIM-3) is newly reported by Monney L in 2002 as an important immune regulatory molecules that can specifically identify Th1 cells from Th2 cells in both mice and human. Engagement of TIM-3 by its ligand galectin-9 negatively regulates IFN-γ secretion and thereby induces T cell tolerance. Accumulated evidences support that TIM-3 is expressed not only on Th1 cells, but also on many other immune cells, such as Tc1, DCs, NK cells and macrophages. Moreover additional Tim-3 ligands were identified, including HMGB1 (high-mobility group box 1) and carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1). The interaction between TIM-3 and ligands induces distinct signalling events which may influence a range of inflammatory conditions, including chronic virus infection, diabetes and multiple sclerosis. In 2007, Wiener Z identified TIM-3 expression on both mast cells and tumor cells in melanoma tissue sections. TIM-3 can be up-regulated by TGF-β treatment and contribute to the lower adhering capacity of tumour cells which may support the survival of melanoma. Emerging evidences show that ectopic TIM-3 expression on lymphoma, NSCLC, clear cell renal cell carcinoma, colon cancer, cervical cancer, etc, was correlated with clinicopathological parameters, and may be used as an independent prognostic factor. Significantly, TIM-3 expression may promote tumor proliferation and metastasis..Our previous work indicated that TIM-3 expression was increased on tumor cells from hepatocellular carcinoma (HCC) patients. HBV and its coding proteins up-regulated TIM-3 expression on hepatoma cell lines. All above strongly suggested that HBV mediated TIM-3 would promote HCC tumorogenesis, metastasis and immune evasion. In this study, we aim to investigate the expression and function of TIM-3 in HCC cells and further explore the mechanism of HBV meidated upreguation of TIM-3. Collectively, TIM-3 can be expressed in solid tumour cells and its expression profile and function might completely contradict in different types of tumors due to distinct biological characteristics. This contradiction strongly suggests a careful caution when TIM-3 is used as an indicator of cancer risk in humans. Thus, it is urgent to explore the expression pattern and function of TIM-3 in different types of tumors. This study was designed to explore the expression and biological functions of TIM-3 in HCC and to identify the novel member of signaling network that is regulated by HBV.

乙型肝炎病毒感染是肝癌发生的重要危险因素，80%的HCC伴有HBV感染，我国属肝癌高发区，揭示HBV相关HCC的发病机制极为重要。TIM-3是表达在T淋巴细胞上的免疫调节分子，与其配体之一galectin-9相互作用诱导免疫耐受。近期文献报道黑色素瘤、淋巴瘤、宫颈癌、非小细胞肺癌等肿瘤细胞上均表达TIM-3，促进癌细胞增殖和迁移，与肿瘤类型及TNM分期相关，可作为肿瘤的独立预后因子，但其在肝癌中的表达及功能尚未见报道。申请者前期在肝癌组织检测到TIM-3异常表达，利用双萤光素酶报告基因系统证实HBV及其编码蛋白可转录激活肝癌细胞中TIM-3的表达，据此提出“HBV转录激活TIM-3促进HCC恶性增殖和免疫逃逸”的假说，拟利用临床标本、细胞模型、小鼠成瘤模型等研究HBV调控肝癌细胞中TIM-3表达的分子机制及在HCC进程中发挥的生物学功能，为进一步揭示HBV感染致癌机制提供良好研究体系。

本课题旨在研究HBV各编码蛋白对宿主基因的调控，寻找受HBV调控的宿主关键基因，并进一步阐明其分子机制，揭示HBV致癌的复杂分子网络调控机制，有望为HCC诊断或治疗提供新切入点。.TIM-3主要表达于Th、巨噬细胞、NK免疫细胞，并异位表达于宫颈癌、非小细胞肺癌、结肠癌等肿瘤细胞, 促进癌细胞增殖和迁移,可作为肿瘤的独立预后因子。本课题在前期工作基础上提出“HBV转录激活TIM-3，进而通过IL-6-pSTAT3通路促进HCC恶性增殖和免疫逃逸”的假说,利用免疫组化、RT-PCR、western-blot、双荧光素酶报告基因检测、流式细胞术、EMSA技术在临床标本、细胞模型、小鼠成瘤模型中研究发现：1.肝癌组织中TIM-3表达水平增高，与HBV呈正相关，其配体HMGB1表达水平相应增高；2.HBV及其编码蛋白尤其是preS2可结合到与TIM-3启动子-183/-153bp位点发挥转录激活作用。3.肝癌细胞过表达TIM-3后IL-6及STAT3磷酸化水平升高，促进细胞增殖。4. TIM-3+肝癌细胞可模拟Th细胞抑制CD8+T淋巴细胞增殖及细胞因子TNF-α and IFN-γ分泌；5.TIM-3可促进小鼠成瘤；本课题以上的结果初步证实TIM-3在肝癌细胞中的表达具有肿瘤特异性，并且受到HBV及其编码蛋白调控，通过IL-6-pSTAT3通路促进肿瘤增殖和免疫逃逸，极有可能是HBV致HCC分子网络中的重要免疫节点，以TIM-3为靶点的诊断、预后评估和治疗手段有望在在临床中应用。