miR-132-RB1/E2F1信号通路与肝癌发生发展的分子流行病学研究

Hepatocellular carcinoma (HCC) is characterized as a cancer with high incidence and mortality and lack of effective systemic therapy in China. Our previous work suggested that miR-132 was probably one of the potential HCC-related microRNAs, and we hypothesized that miR-132 might function as a tumor suppressor via the RB1/E2F1 signaling pathway. In the present study, we will conduct a prospective cohort study in a large number of HCC high-risk subjects chronically infected with HBV. A longitudinal panel comparison of newly diagnosed HCC patients will be performed to examine serum miR-132 pre- and post-paroxysm, and also pre- and post-surgery. Furthermore, a cross-sectional comparison will be utilized to investigate serum miR-132 and HBx mRNA levels among a large number of HCC high-risk subjects consisting of chronic HBV carriers, patients with chronic hepatitis B, liver cirrhosis and HCC, respectively. In addition, the correlation between miR-132 level and HBx mRNA expression will be analyzed. Correlation analysis of miR-132 level and RB1 mRNA/pRB1 expression of serum and tissues in more HCC patients will be conducted. Loss-and gain-of-function experiments will be performed in both HCC cell lines and nude mouse models. The expressions of genes involved in RB1/E2F1 signaling pathway will be assayed post transfection of pre-miR-132/miR-132 mimic/miR-132 inhibitor, and RB1 siRNAs, respectively. The expression pattern of miR-132 and the validation of mechanism involved in RB1/E2F1 signaling pathway in HCC will provide us potential diagnostic and prognostic markers, and afford more epidemiological and experimental basis for non-coding RNAs-based therapy strategy for HCC.

我国肝细胞癌发病率和死亡率高。课题组前期工作表明miR-132可能是肝癌相关microRNA之一，可能通过RB1/E2F1信号通路发挥抑癌作用。本项目将对已建立的大型肝癌高危队列进行前瞻性追踪，纵向比较肝癌发病前后和手术前后血清miR-132表达；横向比较慢性HBV携带者、慢性乙型肝炎、肝硬化和肝癌患者miR-132和HBx mRNA的表达以及二者表达相关性；扩大样本对临床肝癌患者miR-132、RB1在肝癌组织和血清的表达进行关联分析。进一步在肝癌细胞系和裸鼠等动物模型进行功能获得/缺失研究，转染pre-miR-132、miR-132模拟物和抑制物、RB1 siRNAs后观察RB1/E2F1信号通路基因的表达变化，对细胞表型和成瘤的影响。肝癌中miR-132表达模式和RB1/E2F1信号通路机制的验证将为肝癌诊断和判断预后提供分子标志物，为非编码RNA治疗肝癌策略提供流行病学和实验依据。

我国肝细胞癌发病率和死亡率高。课题组前期工作表明miR-132可能是肝癌相关microRNA之一，可能通过RB1/E2F1信号通路发挥抑癌作用。本项目对肝癌中E2F1和miR-132靶基因PIK3IP1相关通路的关键基因及其分子作用机制开展了研究。一方面验证E2F1在肝癌的高表达和评估肝癌患者的预后价值，挖掘相关通路上的中枢基因和靶向基因。结果表明E2F1在肝癌中的表达升高，E2F1在区分肝癌患者和非肝癌对照组具有中等的鉴别能力。E2F1升高与亚洲人种、肿瘤分类、肿瘤组织学分级、ECOG评分和血浆AFP水平相关。E2F1高表达与不良生存状况相关，是肝癌的危险因素。对肝癌差异表达基因、E2F1正相关共表达基因和E2F1预测靶基因的功能富集分析提示细胞周期通路有重要作用，CCNE1和CCNA2是其中的中枢基因。该两基因在肝癌患者中调节细胞周期和促进抗凋亡活性有潜在作用。CCNE1和CCNA2、FEN1、MCM2、MCM4、PRIM2和RNASEH2A在肝癌的高表达与E2F1一致，且与肝癌患者的不良预后相关。(CCNE1:HR=1.7,P=0.0022；CCNA2:HR=1.7,P=0.0041)。进一步对E2F1信号通路相关分子CCNA2和CCNB1表达及其功能分析结果显示：CCNA2、CCNB1在肝癌组织和肝癌细胞中均高表达。上调CCNB1可促进Huh7细胞的增殖，下调CCNB1可抑制Huh7细胞的增殖。CCNB1可能是促进肝癌细胞增殖的重要基因。CCNB1、CCNA2的表达水平与肝癌患者的预后显著相关。另一方面对重新预测的miR-132靶基因RAP2B、PIK3IP1并进行验证，分析PIK3IP1互作蛋白SMARCA4在肝癌的基因和蛋白表达水平，以及SMARCA4表达与肝癌患者临床病理特征的关系。结果显示肝癌组织中SMARCA4 mRNA和蛋白表达水平高于癌旁组织；在女性肝癌患者表达高于男性；在甲胎蛋白（AFP）>2000ng/ml的肝癌患者高于<2000ng/ml患者。以上研究结果为进一步研究miR-132—E2F1/PIK3IP1—SMARCA4的调控通路各相关基因在肝癌发生发展中的作用机制提供了实验基础和理论依据。上述研究基因可能通过调控细胞周期等途径影响肝癌细胞生长、转移和诱导凋亡，从而影响肝癌发生、发展和转归，且对判断肝癌患者预后具有一定的临床参考价值。

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