王金淼,姜　涛,戚　峰,刘　彤，王鹏志（天津医科大学总医院普通外科，天津　300052）



抑制microRNA-21表达降低HCT-116细胞侵袭转移能力的实验研究

王金淼,姜涛,戚峰,刘彤，王鹏志
（天津医科大学总医院普通外科，天津300052）

摘要目的：探讨抑制人结肠癌细胞HCT-116 microRNA-21表达降低结肠癌细胞侵袭转移能力作用机制。方法:应用表达反义microRNA-21的质粒miRZip21转染HCT-116细胞，检测microRNA-21的变化，TargetScan和PicTar等工具筛选与侵袭转移相关的靶基因，实时定量PCR方法检测转染前后靶基因mRNA的变化，Western blot检测靶基因蛋白表达变化，平皿划痕和Transwell实验观察抑制microRNA-21与否HCT-116细胞转移和侵袭能力。结果:筛选出4种与侵袭转移相关的microRNA-21靶基因：TIMP-3，RECK，BMPRⅡ和PCDH17；miRZip21质粒转染HCT-116后，micro-RNA 21表达水平下降约40%，靶基因TIMP-3和RECK的mRNA及靶蛋白表达上升明显，有统计学意义（P <0.05）；平皿划痕和Transwell实验结果表明抑制microRNA-21后，HCT-116细胞转移侵袭能力明显减弱，差异有统计学意义（P <0.05）。结论:抑制microRNA-21的表达能够减弱HCT-116细胞的侵袭和转移能力。

关键词结肠癌；侵袭；转移；microRNA-21；质粒

Experimental research on reducing invasion and metastasis ability of HCT-116 by inhibiting microRNA-21 expression

WANG Jin-miao,JIANG Tao，QI Feng，LIU Tong，WANG Peng-zhi
（Department of General Surgery，General Hospital，Tianjin Medical University，Tianjin 300052，China）

Abstract Objective:To explore the effect on invasion and metastasis of HCT-116 colon cancer cell by downregulating the microRNA-21 expression.Methods:After plasmid miRZip21,miRZipTManti-microRNA-21expression lentivector ,was used to transfect HCT-116,the expression of microRNA-21 was assessed.The microRNA-21 target genes associated with invasion and metastasis were predicted by TargetScan and PicTar.The mRNA and protein of taget genes were detected by qRT-PCR and Western blot respectively.The ability of migration and invasion of HCT-116 after transfection were detected by the Scratch assay and Transwell assay.Results:TIMP-3,RECK,BMPRⅡand PCDH17 were the gene targets associated with invasion and metastasis;After the cells were transfected with plasmid miRZip21，the microRNA-21 level descended to about 60%，the mRNA and protein level of TIMP-3 and RECK increased significantly(P <0.05);Significant reduction of invasion and migration were observed in anti -microRNA -21 -transfected HCT -116(P <0.05).Conclusion:Inhabiting the microRNA-21 expression can lessen the invasive and metastatic ability of HCT-116 cells.

Key words colonic cancer;invasion;metastasis;microRNA-21;plasmid

近年来随着分子生物技术的发展，人们发现结肠癌是一个多步骤、多阶段、多基因参与的遗传性疾病，这些基因参与结肠癌发生、发展、侵袭及转移。microRNA是近年来分子生物学和肿瘤学领域的研究热点，是一类新发现的长度为18～24个核苷酸的内源性非编码单链小分子RNA[1]，成熟的microRNA能够识别特定的目标mRNA,对靶mRNA进行降解或抑制翻译来调节靶基因的表达过程[2]。microRNA-21在多种实体肿瘤细胞均出现异常表达，参与调控多种抑癌基因，提示microRNA-21作为一个致癌microRNA，在肿瘤的发生和发展中发挥着重要作用[3-4]。而当前对结肠癌microRNA-21的研究较少，其对结肠癌转移调控机制尚不清楚，因此通过调节microRNA-21的水平，探讨其在结肠癌转移中的机制，有可能深入了解结肠癌的发生发展过程，为结肠癌的诊断、治疗及预后提供新的思路。……