摘要
目的 探讨微小RNA-429(miR-429)通过调控狭缝引导配体2(SLIT2)对前列腺癌细胞迁移及侵袭的影响.方法 实时荧光定量PCR(qRT-PCR)法测定人前列腺上皮细胞、人前列腺癌细胞22RV1、VCaP、C4-2、DU145、LNCaP、PC-3细胞株中mir-429的表达差异;将22RV1前列腺癌细胞分为空白对照组、miR-429 NC 组、miR-429 inhibitor 组、pcDNA 组、pc-SLIT2 组、miR-429 inhibitor+sh-NC 组和 miR-429 inhibitor+sh-SLIT2 组,测定各组 miR-429 及 SLIT2 的表达,双荧光素酶报告实验检测miR-429与SLIT2的靶向关系,Transwell小室实验检测细胞侵袭能力,划痕实验检测细胞迁移能力,Western blot检测细胞迁移相关蛋白(MMP-2、MMP-9)表达情况.结果 与人前列腺上皮细胞相比,22RV1、VCaP、C4-2、DU145、LNCaP、PC-3细胞中miR-429表达水平显著升高(P＜0.05),其中22RV1前列腺癌细胞miR-429表达水平较高,因此选择22RV1前列腺癌细胞用于后续研究.荧光素酶报告结果显示,SLIT2与miR-429存在靶向关系;与空白对照组相比,miR-429 inhibitor 组、miR-429 inhibitor+sh-SLIT2 组 22RV1 前列腺癌细胞中 miR-429 mRNA表达水平显著降低(P＜0.05),pc-SLIT2组miR-429 mRNA表达水平差异无统计学意义(P＞0.05);miR-429 inhibitor 组、pc-SLIT2 组、miR-429 inhibitor+sh-SLIT2 组细胞侵袭、迁移能力、MMP-2、MMP-9蛋白表达显著降低(P＜0.05),SLIT2蛋白表达显著升高(P＜0.05);与miR-429 inhibitor组相比,miR-429 inhibitor+sh-SLIT2组22RV1前列腺癌细胞侵袭、迁移能力、MMP-2、MMP-9蛋白表达显著升高(P＜0.05).结论 低表达miR-429可抑制22RV1前列腺癌细胞的侵袭及迁移能力,可能是通过上调SLIT2实现的.
Abstract
Objective To investigate the effects of microRNA-429(miR-429)on migration and invasion of prostate cancer cells by regulating SLIT2.Methods The expressions of miR-429 in human prostatic epithelial cells,22RV1,VCaP,C4-2,DU145,LNCaP and PC-3 cell lines were detected by real-time fluorescence quantitative PCR(qRT-PCR),then 22RV1 prostate cancer cells were divided into blank control group,miR-429 NC group,miR-429 inhibitor group,pcDNA group,pc-SLIT2 group,miR-429 inhibitor+sh-NC group and miR-429 inhibitor+sh-SLIT2 group.The ex-pressions of miR-429 and SLIT2 protein were detected,and dual luciferase reporter assay was used to detect the targeting relationship between miR-429 and SLIT2.Transwell chamber assay was used to detect the invasion ability and scratch test was used to detect the migration ability,Western blot was used to detect the expressions of MMP-2 and MMP-9.Results Compared with those in hu-man prostatic epithelial cells,the expression levels of miR-429 in 22RV1,VCaP,C4-2,DU145,LNCaP and PC-3 cells significantly increased(P＜0.05).The expression level of miR-429 was high-er in 22RV1 prostate cancer cells,therefore,22RV1 prostate cancer cells were selected for follow-up study.Luciferase assay showed the targeting relationship between miR-429 and SLIT2.Compared with the blank control group,the expression levels of miR-429 mRNA in 22RV1 prostate cancer cells of miR-429 inhibitor group and miR-429 inhibi-tor+sh-SLIT2 group significantly decreased(P＜0.05),while there was no significant difference in miR-429 mRNA expres-sion in pc-SLIT2 group(P＞0.05).Compared with the blank control group,miR-429 inhibitor group,pc-SLIT2 group and miR-429 inhibitor+sh-SLIT2 group had significantly lower cell invasion and migration abilities and expressions of MMP-2 and MMP-9 proteins in 22RV1 prostate cancer cells,and significantly higher expression of SLIT2 protein(P＜0.05).Compared with miR-429 inhibitor group,miR-429 inhibitor+sh-SLIT2 group had significantly higher cell invasion,migration abilities and expressions of MMP-2 and MMP-9 proteins in 22RV1 prostate cancer cells(P＜0.05).Conclusions Low expression of miR-429 can inhibit the invasion and migration of 22RV1 prostate cancer cells,and it may be achieved by up-regulating SLIT2.
维普
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