HBV感染诱导表达的novel-miR-12对肝癌细胞恶性行为的影响及机制研究

In recent years, increasing evidence have showed that aberrant expression of microRNAs (miRNAs) by HBV infection are implicated in the pathogenesis of hepatocellular carcinoma (HCC). In our study, we have identified that a novel miRNA (novel-miR-12) is highly expressed in HBV-positive HCC tissues. We also determined the effects of novel-miR-12 on the malignant behaviors of HCC cells in vitro. We found that novel-miR-12 promoted HCC cell growth, migration and invasion capacity, and the transformation ability of normal immortalized hepatocytes. All these results indicated that novel-miR-12 functioned as an oncogene in HCC. Furthermore, we indicated that novel-miR-12 directly targeted and up-regulated BAMBI. Functional experiments showed that BAMBI could promote HCC cell growth, and might enhance the malignant transformation of normal liver cells. On these bases, we will further investigate the effect of novel-miR-12 on tumorigenicity and metastasis of HCC cells in vivo，and the role of BAMBI in HCC cells. Novel-miR-12 may promote HCC development by affecting multiple target genes. Therefore, other candidate target genes need to be identified. Moreover, we will explore the role of the identified targets and confirm that the phenotype caused by novel-miR-12 is mediated by modulating target genes using rescue experiments. In addition, the upstream transcription factor that regulates the expression of novel-miR-12 will be identified, and the key element of HBV that activates the transcription factor will be determined. All of these will be helpful to elucidate the regulation mechanism of the abnormal expression of novel-miR-12 in HBV related HCC and to identify the downstream molecule mechanisms through which novel-miR-12 affects the activities of HCC cells.

目前研究表明HBV感染诱导的miRNAs参与了肝癌的发生发展过程。本项目前期研究发现一个在HBV阳性肝癌细胞中异常高表达的miRNA分子novel-miR-12，并证实其促进肝癌细胞体外生长、迁移和侵袭，以及正常肝细胞的恶性转化能力，从而发挥促癌基因作用。进一步筛选确定BAMBI是novel-miR-12的直接靶基因，且其能够促进肝癌细胞体外生长和正常肝细胞的恶性转化。在此基础上，我们拟进一步评价novel-miR-12对肝癌细胞体内成瘤和转移能力的影响，并完善BAMBI在肝癌细胞中的功能研究。Novel-miR-12可能通过影响多个靶基因来实现其促癌基因作用，所以我们拟进一步鉴定并探究其他的候选靶基因。此外，确定调控novel-miR-12表达的上游因子，并解析HBV感染改变转录调控的分子机制，从而深入阐明novel-miR-12在HBV相关肝癌中异常表达的上游调控机制及其下游作用机制。

本项目鉴定出一个新的在HBV阳性肝癌组织中异常高表达的miRNA分子—novel-miR-12（缩写为miR-HCC2），功能学实验表明，miR-HCC2促进肝癌细胞体内外生长、迁移、侵袭以及干细胞样特性。进一步筛选鉴定发现miR-HCC2通过靶定BAMBI加速肝癌细胞周期进程从而促进肝癌细胞的生长，以及通过靶定ELMO1，从而促进EMT进程增强肝癌细胞迁移与侵袭能力。本研究还发现miR-HCC2可以通过靶定BAMBI，从而激活Wnt/β-Catenin信号通路，促进肝癌细胞的干细胞特异性转录因子的表达、CD90+细胞比例与细胞成球能力，提示其参与调控肝癌细胞的干细胞样特性。本研究进一步锁定了miR-HCC2的两个核心启动子片段，并鉴定出结合核心启动子从而促进miR-HCC2高表达的上游转录因子YY1，揭示了miR-HCC2在HBV阳性肝癌组织中异常高表达的上游调控机制。此外，本研究表明miR-HCC2可以反向调控HBV的复制与转录，其可能为病毒慢性感染提供了条件，为HBV长期潜伏感染宿主细胞提供了新的机制。. 本项目成果如下：.（1）发现与鉴定了一个在HBV阳性肝癌组织中异常高表达的miRNA分子-miR-HCC2，并阐明了miR-HCC2在HBV相关肝癌发生发展中的具体作用机制。MiR-HCC2通过靶定BAMBI和ELMO1，从而促进肝癌细胞体内外生长和转移能力，并增强正常肝细胞的恶性转化能力，发挥其促癌基因作用。 .（3）揭示了miR-HCC2参与调控肝癌细胞干细胞样特性的分子机制。MiR-HCC2通过靶定BAMBI，从而激活Wnt/β-Catenin信号通路，促进肝癌细胞的干细胞特异性转录因子的表达、CD90+细胞比例与细胞成球能力，为理解HBV感染诱导肝癌的转移和复发提供新的认识。.（4）明确了YY1为结合miR-HCC2核心启动子的上游转录因子，HBV通过上调转录因子YY1，从而激活miR-HCC2的表达，揭示了miR-HCC2在HBV阳性肝癌组织中异常高表达的上游调控机制。.（5）解析了miR-HCC2在调控HBV复制与基因表达中的作用，miR-HCC2通过抑制HBV的EnhancerI/Xp启动子的活性，从而抑制HBV的转录与抗原表达，其可能为病毒慢性感染提供了条件，揭示了HBV长期潜伏感染宿主细胞的新机制。