P2-HNF4α促进肝癌细胞增殖的分子机制

Hepatocellular carcinoma (HCC), with high morbidity and mortality, is one of the most prevalent malignancies worldwide. However, the nature of HCC progression remains elusive. It has been demonstrated that P1 promoter-driven HNF4α (P1) plays an important role in HCC cell proliferation. Recently, we found that expression of P2 promoter-driven HNF4α (P2) was increased in HCC, and inversely associated with P1 expression. P2 expression seemed to be modulated by promoter methylation. Statistical analysis revealed that P2 expression was correlated with tumor differentiation and prognosis. Following study showed that knockdown of P2 expression inhibited HCC cell viabilities. Based on our findings, we assume that P2 functions differently from P1, and promotes cell proliferation in HCC. In this study, we aim, in vitro and in vivo, to determine P2 expression and its clinical significance in HCC, to disclose the regulation of P2 expression, to quest for the pathway via which P2 promotes cell proliferation, to reveal the mechanism through which P2 is involved in tumor growth and differentiation, using a series of biological and immunological experiments in cell and animal models. Our study is likely to provide not only solid evidence that P2 is capable of facilitating cell proliferation and differentiation, but also promising therapeutic target in HCC treatment.

肝细胞癌恶性度高，预后差，其发生发展机制尚未明了。已有研究表明P1启动的HNF4α（P1）在抑制肿瘤细胞增殖中有重要作用。我们前期观察到P2启动的HNF4α（P2）在肝癌细胞中高表达，且与P1表达负相关，并受启动子甲基化修饰调控；统计分析提示P2表达与肝癌分化程度及预后相关；实验表明沉默P2表达抑制肝癌细胞增殖。因此，我们提出P2与P1功能不同，并促进肝癌细胞增殖的假说。本项目拟采用一系列分子细胞生物学及免疫学实验技术，利用过表达或敲除P2的细胞和动物模型，在蛋白表达调控及细胞功能上，明确P2在肝癌中的表达及其临床意义，揭示P2在肝癌中高表达的调控机制，寻找P2促进细胞增殖的作用通路，探讨P2在肝癌细胞生长中的作用及其分子机制，并阐明P2在肝癌细胞分化中的作用，从而获得P2参与肝癌细胞增殖和分化的可靠证据，为诠释肝癌发生发展的分子机制和寻找肝癌治疗新靶点提供充分的科学依据。

肝细胞癌恶性度高，预后差，其发生发展机制尚未明了。已有研究表明由不同启动子介导的不同基因转录本在肿瘤恶性进展中可能发挥重要作用。文献报道HNF4α存在两个启动子：P1和P2。由P1启动子启动的HNF4α (P1)在抑制肿瘤细胞增殖中有重要作用，但是，我们采用qRT-PCR, western blot, IHC等技术在615例肝癌样本中的研究结果提示由P2启动子启动的HNF4α (P2)在肝癌细胞中高表达，与P1表达负相关；统计分析提示P2表达与肝癌分化程度及预后相关。在研究P2生物学功能时，我们利用裸鼠模型，在体内检测P2是否能促进肿瘤生长。实验顺利开展，但实验结果并未能符合实验预期结果：在多次的体内外实验中，过表达P2或者沉默P2表达未能影响肝癌细胞的克隆形成能力、细胞迁移及体内成瘤能力。因此，课题组决定修正项目研究内容，转向寻找鉴定与肝癌发生发展密切相关的其他分子标记物。通过8对肝癌及其癌旁组织进行转录组测序分析，我们鉴定并验证出Zic2, AIM2, LRG1, PKM2, Bad, miR-137等多个基因在肝癌中表达异常，并阐述这些基因在肝癌中的表达，意义，生物学功能及其作用机制。在本项目的资助下，课题组共发表SCI论文10篇，总影响因子45.26分，单篇最高6.375分；参加学术交流活动21人次；共培养博士研究生7名，硕士研究生1名。综上所述，课题组能基本完成原研究内容，在高水平SCI杂志上发表了数篇文章，达到了部分既定研究目标。