Identification of differentially expressed miRNAs in HCC cell lines compared with normal livers

To explore functionally crucial tumor-suppressive (TS)-miRNAs in hepatocellular carcinoma (HCC), we performed integrative function- and expression-based screenings of TS-miRNAs in six HCC cell lines. The screenings identified seven miRNAs, which showed growth-suppressive activities through the overexpression of each miRNA and were endogenously downregulated in HCC cell lines. Further expression analyses using a large panel of HCC cell lines and primary tumors demonstrated four miRNAs, miR-101, -195, -378 and -497, as candidate TS-miRNAs frequently silenced in HCCs. Among them, two clustered miRNAs miR-195 and miR-497 showed significant growth-suppressive activity with induction of G1 arrest. Comprehensive exploration of their targets using Argonute2-immunoprecipitation-deep-sequencing (Ago2-IP-seq) and genome-wide expression profiling after their overexpression, successfully identified a set of cell-cycle regulators, including CCNE1, CDC25A, CCND3, CDK4, and BTRC. Our results suggest the molecular pathway regulating cell cycle progression to be integrally altered by downregulation of miR-195 and miR-497 expression, leading to aberrant cell proliferation in hepatocarcinogenesis. Screening of frequently downregulated miRNAs by comparing endgeneous expression status of miRNAs in 6 HCC cell lines with 2 normal livers Expression analysis using total RNAs extracted from standard medium conditioned 6 HCC cell lines, and 2 normal livers derived from patients with hepatectomy due to metastatic liver tumor

为探索肝细胞癌（hepatocellular carcinoma, HCC）中功能至关重要的抑癌miRNA（tumor-suppressive miRNA, TS-miRNA），我们针对6株肝癌细胞系开展了基于功能与表达的抑癌miRNA整合筛选。
本次筛选共鉴定出7株miRNA，这些miRNA通过单株过表达即可发挥生长抑制活性，且在内源性表达层面于肝癌细胞系中呈下调状态。
后续我们利用大量肝癌细胞系与原发性肿瘤样本开展进一步表达分析，最终确定miR-101、miR-195、miR-378及miR-497这4株miRNA为肝癌中频繁发生沉默的候选抑癌miRNA。
其中，成簇存在的miR-195与miR-497可通过诱导G1期阻滞，展现出显著的生长抑制活性。
我们通过Argonaute2免疫共沉淀深度测序（Argonute2-immunoprecipitation-deep-sequencing, Ago2-IP-seq），以及二者过表达后的全基因组表达谱分析，对其靶基因进行了全面探索，成功鉴定出包括CCNE1、CDC25A、CCND3、CDK4与BTRC在内的一系列细胞周期调控因子。
本研究结果表明，miR-195与miR-497的表达下调可整体性调控细胞周期进程的分子通路，最终导致肝细胞癌变过程中出现异常细胞增殖。
本研究的筛选流程为：通过对比6株肝癌细胞系与2例正常肝组织的miRNA内源性表达水平，筛选在肝癌中频繁下调的miRNA；
表达分析流程为：采用从标准培养基培养的6株肝癌细胞系，以及因转移性肝肿瘤接受肝切除术的患者来源的2例正常肝组织中提取的总RNA开展实验。