miR100-FZD8-β-catenin轴在环境因素促发原发性肝癌过程中的关键作用及机制

Liver cancer has become a serious health problem in human due to its ever-increasing incidence and mortality, but the underlying molecular mechanisms remain elusive. Various environmental factors has been reported to contribute to liver cancer, in which, however, the key signalling needs to be further explored. Our previous study revealed that miRNA plays an essential role in the carcinogenesis induced by environmental factors in coordination with genetic mutation. More specifically, miR100 inactivation is involved hepatocarcinogenesis induced by arsenic or alcohol consumption. Environmental factors-induced miR100 inactivation and miR100 knockdown both promote malignant transformation of hepatocyte, possibly by targeting Frizzled-8 (FZD8)-mediated glycogen synthase kinase-3β (GSK-3β) phosphorylation (pGSK-3β) and nuclear translocation of β-catenin. Moreover, Inverse relation of miR100 expression and pGSK-3β level in liver cancer demonstrated in our preliminary data, as well as the aberrant activation of Wnt signalling based on the literature, strongly indicate that miR100-FZD8-β-catenin signal axon may be play a key role in hepatocarcinogenesis promoted by environmental factors. In this proposal, we will validate our central hypothesis by using our established miR100 conditional knock-out mouse and cellular model in combination with clinical sample analysis, with objectives to investigate the impact of miR100-FZD8-β-catenin axon on hepatocarcinogenesis, and to elucidate the underlying molecular mechanisms. We expect that the results from this study will provide new clues for the prevention and treatment of the deadly disease of liver cancer.

肝癌因发病率和死亡率居高不下严重危害人类健康，且机制不清。已发现多种环境因素可诱导基因突变等促发肝癌，但对其中的关键信号调控认识不深。本组前期研究发现miRNA在环境因素与基因突变协同致癌过程中发挥重要作用，其中miR100失活可能参与了砷、酒精等环境因素诱发的小鼠肝癌发生；环境因素可诱导肝细胞miR100失活，miR100敲低能加速肝细胞恶性转化，且miR100可能靶向FZD8促进GSK-3β磷酸化及β-catenin入核。结合肝癌组织miR100表达降低、pGSK-3β升高的预实验结果及肝癌细胞中Wnt信号异常活化的文献报道，我们认为miR100-FZD8-β-catenin信号轴可能是环境因素促发肝癌的关键环节之一。本课题拟用已建立的miR100敲除小鼠及细胞模型，结合临床样本，研究miR100-FZD8-β-catenin轴在肝癌发生中的作用，并阐明其机制，为肝癌的防治提供新线索。

肝癌因发病率和死亡率居高不下严重危害人类健康，且机制不清。前期研究结果表明miR100在肝癌样本中低表达可能参与肝癌发生，但其分子机制不明。本课题通过遗传学手段以及化学干预的方法，首先建立miR100低表达的肝细胞以及肝脏特异性敲除miR100的小鼠。通过生物学实验方法检测miR100缺失的衰老小鼠（15个月）肝脏细胞呈现明显的病理性改变，如炎性细胞浸润以及肝细胞核增大。并且肝功能异常升高，肝细胞代谢方式趋向于糖酵解。已经有文献报道肝炎病毒感染可以诱导肝脏 miR100 表达下调，miR100在包括肝癌在内的多种肿瘤组织中表达下调，尤其在肿瘤干细胞中表达更低。我们有理由推测： miR100缺失可能是多种环境因素（例如HBX过表达）促发肝癌的共同机制之一。结果显示HBX协同miR100缺失体内与体外同时促进STAT3信号通路活化并进而促进正常肝细胞发生恶性转化。既然HBX协同miR100缺失可促进肝癌发生，并且与STAT3信号通路相关。另外miR100缺失同样可以促进DEN诱导的肝癌发生。并且同样可以刺激STAT3信号通路的活化。HGF/c-Met信号通路，以及与此通路紧密相关的ERK,AKT, mTOR等途径在肝癌的发生发展中扮演着重要的角色。因此，本课题组试图从源头设计能竞争结合HGF的新药，其中，我们发现C7肽段最为理想。为了进一步验证其在肝癌治疗中的作用，本课题组在体外水平验证其对肝癌细胞的恶性生物学影响。结果发现，C7肽段可以抑制HGF依赖的肝癌细胞转移和侵袭能力。随后，本课题组深入分析了C7肽段抑制肝癌细胞恶性的机制。结果显示，C7肽段可以抑制MET，AKT和ERK信号通路，并且这一效应是浓度依赖的。体内实验验证了C7肽段确实可以减缓肝癌的转移，并且可以抑制MET和ERK信号通路。另外，miR100参与调控其他肿瘤或者生物学进程。例如，在宫颈癌细胞中，一旦敲低miR-100,宫颈癌细胞的增殖，转移侵袭能力都有显著提高。并且，miR-100相关的SHP2,AKT, ERK, mTOR信号通路都有不同程度的活化。miR100缺失小鼠的外周血的髓系细胞比例升高。进一步研究发现，miR100缺失可调控MOSPD2的表达，进而促进髓系细胞迁移。此课题揭示了miR100参与肝癌发生的分子机制，并且为肝癌的治疗提供新的视角。