miR-202通过降低肝癌细胞ROCK1表达抑制其迁移和侵袭

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摘要目的：研究微小RNA-202（miR-202）对肝癌细胞迁移和侵袭能力的影响，并探讨其可能的分子机制。方法：通过实时定量PCR检测Hep3B、Huh7及HCCLM3人肝癌细胞和LO2人正常肝细胞中miR-202的表达水平。以miR-202表达水平最低的HCCLM3细胞为转染对象，转染miR-202模拟物或空白对照miRNA，以miR-202表达水平最高的Hep3B细胞为转染对象，转染miR-202抑制物或阴性对照miRNA，并利用实时定量PCR验证转染效率。采用Transwell迁移和侵袭实验检测过表达和敲低miR-202对肝癌细胞迁移和侵袭能力的影响。生物信息学网站检索并应用荧光素酶报告基因实验验证miR-202的靶基因。实时定量PCR和Western blot检测miR-202靶基因的表达水平。结果：与正常肝细胞LO2相比，miR-202在肝癌细胞的表达水平显著降低；HCCLM3细胞转染miR-202模拟物后，细胞miR-202的表达水平显著增加；过表达miR-202后，HCCLM3细胞的迁移和侵袭能力显著降低；Hep3B细胞转染miR-202抑制物后，细胞miR-202的表达水平显著降低；敲低miR-202后，Hep3B细胞的迁移和侵袭能力显著增强；生物信息学检索及荧光素酶报告基因证明ROCK1基因为miR-202的靶基因。过表达miR-202可明显降低肝癌细胞中ROCK1基因的表达，而敲低miR-202可明显增加肝癌细胞中ROCK1基因的表达。结论：miR-202在肝癌细胞中低表达，miR-202通过抑制ROCK1的表达抑制肝癌细胞的迁移和侵袭。

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	袁琳琳
	喻晓芬

关键词 ： , miR-202, 肝癌, ROCK1, 迁移, 侵袭

Abstract：Objective: To observe the effect of microRNA-202 on the migration and invasion of hepatocellular carcinoma (HCC) cells and explore the possible molecular mechanism. Methods: The expression level of miR-202 in human HCC cells (Hep3B, Huh7, HCCLM3) and human normal hepatic cells (LO2) was detected by quantitative real-time PCR (qRT-PCR). miR-202 mimic or control microRNA (miR-control) was transfected into the HCCLM3 cells and miR-202 inhibitor or negative control microRNA (miR-NC) was transfected into the Hep3B cells. Transfection efficiency was examined by qRT-PCR. The effect of miR-202 on the migration and invasion of HCC cells was detected by Transwell assay. Bioinformatics website TargetScan was used to predict the potential targets of miR-202 and dual luciferase reporter assay was used to validate the target gene of miR-202. qRT-PCR and Western blot were used to detect the expression of miR-202 target gene. Results: The expression of miR-202 in normal liver cells was significantly higher than that in HCC cells. Transfection of miR-202 mimics significantly increased the expression level of miR-202 in HCCLM3 cells, and led to the decreased ability of cell migration and invasion. Transfection of miR-202 inhibitor significantly decreased the expression level of miR-202 in Hep3B cells, and led to the increased ability of cell migration and invasion. Bioinformatics website and dual luciferase reporter gene assay confirmed ROCK1 was the target gene of miR-202. Overexpression of miR-202 significantly reduced the expression of ROCK1 while miR-202 knockdown led to increased expression of ROCK1. Conclusion: The expression of miR-202 is down-regulated in HCC cells. miR-202 inhibits the migration and invasion of HCC cells by down-regulating the expression of ROCK1 gene.

Key words： MicroRNA-202 liver cancer ROCK1 migration invasion

收稿日期: 2018-09-24

基金资助:浙江省公益性技术应用研究计划项目（2015C33153）。

通讯作者: 喻晓芬，主任护师，Email：yxf4800@163.com

作者简介: 袁琳琳（1981-），女，浙江杭州人，主管护师。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2019/V49/I3/204

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