肝癌潜在标志物CCL15在乙肝病毒X蛋白促肝癌侵袭和转移中的分子机制及其临床检测价值

Our study found that chemokines CCL15 was significantly increased in serum and tissues of patients with HCC related to HBV. CCL15 had high sensitivity and specificity in the diagnosis for liver cancer. The expressions of CCL15 in serum and tissues were positively correlated with metastatic indexes of liver cancer. CCL15 had a low survival rate and was closely related to the prognosis of liver cancer. The overexpression of CCL15 promoted the migration and invasion of liver cancer cells and its down-expression to reduce the migration and invasion of HCC cells. It was found that HBx significantly increased CCL15 expression. This project will be based on the previous research further in-depth discussion that HBx regulates CCL15, and to elucidate the molecular mechanism, the main research contents are: to further confirm diagnosis and prognostic value of CCL15 for liver cancer by enlarging the serum and tissue samples of liver cancer, the application of molecular biology methods in molecular, cellular, animal and clinical specimens and other aspects detailed study the regulation of CCL15 by HBx; The role of CCL15 in promoting the migration and invasion of hepatocellular carcinoma cells is observed through the experiments of animal tumors, cell lines and transwell. Through the effects of transcription factors, microRNA and its CCL15 signal pathway network, the molecular mechanism of regulation of CCL15 by HBx for the migration and invasion of HCC cells will be emphasized. The study will further enrich the molecular mechanisms of HBx for cancer metastasis and provide a new target or potential tumor marker for the treatment and diagnosis of liver cancer.

我们前期研究发现：①CCL15在肝癌血清诊断中有较高的敏感性和特异性，血清及组织中CCL15高表达与肝癌转移及预后不良密切相关；②CCL15过表达促进肝癌细胞迁移和侵袭，抑制其表达降低肝癌细胞迁移和侵袭能力；③已筛选发现HBx显著上调CCL15表达。本项目将在前期研究基础上进一步深入探讨HBx 对CCL15的调节作用，并阐明其分子机制及临床检测价值，主要研究内容有：扩大肝癌血清、组织样本进一步确认CCL15对肝癌的诊断及预后判断价值，应用分子生物学手段在分子、细胞、动物和临床标本等层面详细研究HBx对CCL15的调节作用；通过动物成瘤、细胞划痕和transwell等实验，观察CCL15在HBx促进肝癌细胞迁移和侵袭中的作用；通过转录因子、microRNA及其CCL15信号通路网络的影响，重点阐明HBx调节CCL15促肝癌细胞侵袭和转移的分子机制，为治疗肝癌提供新的靶点和潜在肿瘤标志物。

乙型肝炎病毒（hepatitis B virus，HBV）是导致肝脏疾病和肿瘤的主要原因之一。HBV病毒X区是最小的开放阅读框，是HBV基因组中结构和功能重叠最明显的区域，X区编码的蛋白称为乙肝病毒X蛋白(HBx)，HBx近年来已成为研究肝脏疾病和肿瘤发病机制的热点之一。本项目应用分子生物学手段在分子、细胞、动物和临床标本等层面详细探讨HBx 对CCL15的调节作用，并阐明其分子机制及临床意义。在HBV阳性的肝癌组织中通过qRT-PCR检测，趋化因子CCL15mRNA在肝癌组织中的表达明显高于对应的癌旁组织，同时研究结果显示HBV阳性的肝癌组织中CCL15 mRNA与HBx mRNA表达存在着明显正相关性；通过western blot 显示HBV阳性的肝癌组织中CCL15和HBx蛋白表达水平均高于对应的癌旁组织；通过免疫组化同样显示在HBV阳性的肝癌组织中HBx与CCL15表达存在着正相关性。细胞实验证实在正常肝细胞及肝癌细胞中过表达HBx后能够显著促进CCL15的表达及分泌，降表达HBx后CCL15表达减少；通过对CCL15启动子序列进行截短证实HBx通过作用于CCL15 0-250bp启动子而促进其转录，通过对CCL15 0-250bp启动子进一步分析发现HBx可以通过p53来调控CCL15的转录，从而明确HBx能够通过p53来调控CCL15的表达；体内及体外实验证实SMAD4作为CCL15另一个重要的调控因子能够促进肝癌细胞的增殖，并且受HBx蛋白的调控；通过进一步分子调控机制研究显示HBx可以通过转录因子TFII-I来作用于SMAD4启动子从而促进其转录、同时还发现HBx蛋白可以结合SMAD4蛋白而抑制其发生泛素化降解促进SMAD4蛋白的表达。本项目将进一步丰富HBx促癌转移的分子机制，为肝癌治疗提供新的靶点、为肝癌诊断和预后判断提供新的肝癌潜在标志物。