抑制microRNA-21表达降低HCT-116细胞侵袭转移能力的实验研究

摘要

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, BMPRⅡ and 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.%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。