微RNA-21在肝细胞癌中的表达及其靶基因研究

目的 探讨微RNA(miRNA)-21在肝细胞癌中的表达特征及其功能,筛选并鉴定miRNA-21可能调控的靶基因.方法 用PAGE/Northern blot方法分析miRNA-21在10例肝细胞癌患者组织标本中的表达特征,并在3种肝癌细胞株中加以验证.用生物信息学分析预测miRNA-21可能的靶基因,通过逆转录-聚合酶链反应(RT-PCR)方法克隆靶基因的调控区序列,利用荧光素酶双报告基因系统筛选,并通过Western blot、流式细胞仪在蛋白质水平进行分析验证.用体外细胞侵袭实验分析miRNA-21与肝癌细胞侵袭能力的关系.结果 miRNA-21在10例肝细胞癌患者中均表达上调,与癌旁组织比较,表达量增加10倍以上.在3种肝癌细胞株中也得到相似的结果.筛选并确认了RECK为miRNA-21的调控靶基因之一.抑制miRNA-21的表达可明显降低肝癌细胞的侵袭.结论 miRNA-21在肝细胞癌中表达失控,通过抑制RECK表达促进肿瘤细胞侵袭.

Expression and function research of miRNA-21 in hepatocellular carcinoma

Objective To research the expression signature and function of miRNA-21 in hepatocellular carcinoma (HCC), and indentify its functional target mRNA.Methods The expression signature of miRNA-21 in 10 cases of HCC was confirmed in 3 kinds of HCC cell lines by PAGE/Northern blot.The potential target mRNA prediction of miRNA-21 was performed by bioinformatics.The regnlation region of miRNA-21 in RECK mRNA was cloned by RT-PCR, and confirmed by sequencing.Dual report gene assay was used to identify the target gene of miRNA-21.The target gene was confirmed by Western blot and FCM analysis.The effect of miRNA-21 on HCC cell line HepG2 was analyzed by in vitro invasion assay.Results miRNA-21 was over-expressed in 10 cases of HCC and 3 kinds of cell lines.RECK was identified as one of miRNA-21 target genes.The invasion ability of HepG2 was suppressed when miRNA-21 was knocked down.Conclusion miRNA-21 was over-expressed in HCC, and promoted cancer cell invasion by inhibiting the expression level of RECK.