miR-4429通过MTDH抑制前列腺癌细胞增殖、迁移与侵袭

doi:10.3969/j.issn.1673-8640.2021.012.015

摘要/Abstract

摘要：

目的探讨微小RNA-4429（miR-4429）对前列腺癌细胞增殖、迁移与侵袭的影响及其调控机制。方法将miR-4429模拟物（miR-4429 mimic）、miR-4429抑制物（miR-4429 inhibitor）、模拟物阴性对照（mimic NC）和抑制物阴性对照（inhibitor NC）质粒转染至前列腺癌细胞系DU145、22rv1、PC3、LNCaP、VCaP和人前列腺上皮细胞系RWPE-1中,采用实时荧光定量聚合酶链反应（RT-qPCR）检测miR-4429的相对表达量。采用CCK-8法、集落形成试验、Transwell小室法和基质胶侵袭试验检测miR-4429对PC3细胞增殖、迁移和侵袭的影响。采用生物信息学方法预测miR-4429可能作用的靶基因。通过荧光素酶报告基因试验确定miR-4429和异黏蛋白（MTDH）是否结合。采用RT-qPCR和免疫印迹法检测miR-4429对MTDH mRNA和蛋白表达的影响。结果 5种前列腺癌细胞系miR-4429相对表达量均显著低于人前列腺上皮细胞系RWPE-1（P<0.05）,其中PC3细胞最低（P<0.01）。miR-4429 mimic组miR-4429相对表达量显著高于mimic NC组（P<0.01）,克隆形成、迁移及侵袭细胞数目明显低于mimic NC组（P<0.01）。miR-4429 inhibitor组miR-4429相对表达量显著低于inhibitor NC组（P<0.01）,克隆形成、迁移及侵袭细胞数目显著高于inhibitor NC组（P<0.01）。荧光素酶报告基因试验结果证实miR-4429和MTDH靶向结合。与RWPE-1细胞相比,PC3细胞中MTDH mRNA和蛋白相对表达量显著升高（P<0.01）。转染miR-4429 mimic后,PC3细胞中MTDH mRNA和蛋白相对表达量明显低于mimic NC组（P<0.01）。转染miR-4429 inhibitor后,PC3细胞中MTDH mRNA和蛋白相对表达量明显高于inhibitor NC组（P<0.01）。过表达MTDH可显著提高PC3细胞的增殖、迁移和侵袭能力,且可逆转miR-4429对PC3细胞增殖、迁移和侵袭能力的抑制作用。结论 miR-4429能够抑制PC3细胞的增殖、迁移及侵袭能力,其机制与下调MTDH基因的表达有关。

关键词: 微小RNA-4429, 前列腺癌, 增殖, 迁移, 侵袭

Abstract:

Objective To study the effects of microRNA（miR）-4429 on the proliferation,migration and invasion of prostate cancer cells,and to investigate the potential related regulatory mechanism. Methods Real-time fluorescence quantitative polymerase chain reaction（RT-qPCR）was used to determine the relative expression of miR-4429 in prostate cancer cell lines（DU145,22rv1,PC3,LNCaP,VCaP） and human prostate epithelial cells（RWPE-1）. The subjects were classified into miR-4429 mimic group,miR-4429 inhibitor group,mimic negative control（NC） group and inhibitor NC group. The effects of miR-4429 on proliferation,migration and invasion of PC3 cells were determined by CCK-8,colony formation test,Transwell chamber test and matrix gel invasion test. The possible target genes of miR-4429 were predicted by bioinformatics analysis,and the binding of miR-4429 and metadherin（MTDH）was determined by luciferase reporter assay. The effects of miR-4429 on MTDH mRNA and protein levels were determined by RT-qPCR and western blotting. Results The relative expression of miR-4429 in 5 prostate cancer cell lines was lower than that in normal human prostate epithelial cells（RWPE-1）（P<0.05）,especially in PC3 cells（P<0.01）. After transfection with miR-4429 mimic,the relative expression of miR-4429 was higher than that in mimic NC group（P<0.01）,and the numbers of clone formation,migration and invasion cells were lower than those in mimic NC group（P<0.01）. After transfection with miR-4429 inhibitor,the relative expression of miR-4429 was lower than that in inhibitor NC group（P<0.01）,and the numbers of clone formation,migration and invasion cells were higher than that in inhibitor NC group（P<0.01）. The binding sites of miR-4429 and MTDH were confirmed by luciferase reporter assay. Compared with RWPE-1,the relative expressions of MTDH mRNA and protein in PC3 cells were increased（P<0.01）. The relative expressions of MTDH mRNA and protein in miR-4429 mimic group were lower than those in mimic NC group（P<0.01）. The relative expressions of MTDH mRNA and protein in PC3 cells transfected with miR-4429 inhibitor were higher than those in inhibitor NC group（P<0.01）. The proliferation,migration and invasion abilities of PC3 cells were promoted by MTDH overexpression. MTDH overexpression reversed the inhibitory effect of miR-4429 on proliferation,migration and invasion of PC3 cells. Conclusions Inhibit miR-4429 could inhibit the proliferation,migration and invasion of PC3 cells,and its mechanism is the down-regulation of MTDH.

Key words: MicroRNA-4429, Prostate cancer, Proliferation, Migration, Invasion

中图分类号:

R446.1

俞莹, 李建杰, 李汉华, 黄娟, 翁文浩, 陈学飞. miR-4429通过MTDH抑制前列腺癌细胞增殖、迁移与侵袭[J]. 检验医学, 2021, 36(12): 1267-1273.

YU Ying, LI Jianjie, LI Hanhua, HUANG Juan, WENG Wenhao, CHEN Xuefei. Inhibit miR-4429 proliferation,migration and invasion of prostate cancer cells by targeting metadherin[J]. Laboratory Medicine, 2021, 36(12): 1267-1273.

图/表 6

图1 不同人前列腺癌细胞系和人前列腺上皮细胞系RWPE-1中miR-4429的相对表达量注：与RWPE-1细胞比较,*P<0.05、**P<0.01。

图2 各组miR-4429相对表达量比较

图3 miR-4429对PC3细胞增殖、迁移和侵袭能力的影响注：（a）CCK-8细胞增殖试验; ▲ miR-4429 inhibitor组; ▲ inhibitor NC组; ■ miR-4429 mimic组; ● mimic NC组;与inhibitor NC组比较,*P<0.01;与mimic NC组比,#P<0.01;（b）细胞克隆试验;（c）迁移试验;（d）各组迁移细胞数目比较;（e）侵袭试验;（f）各组侵袭细胞数目比较。

图4 miR-4429与MTDH的结合点及荧光素酶报告基因试验结果注：（a）miR-4429与MTDH 3'UTR区的结合序列;（b）荧光素酶报告基因试验; mimic NC组; miR-4429 mimic组; inhibitor NC组; miR4420 inhibitor组。

图5 miR-4429对MTDH mRNA和MTDH蛋白表达的影响注：（a）RWPE-1和PC3细胞中MTDH mRNA的相对表达量;（b）RWPE-1和PC3细胞中MTDH蛋白表达的免疫印迹图;（c）各组MTDH mRNA的相对表达量;（d）各组MTDH蛋白表达的免疫印迹图。

图6 过表达miR-4429或MTDH对PC3细胞增殖,迁移和侵袭的影响注：（a）过表达miR-4429或MTDH对PC3细胞增殖的影响;（b）过表达miR-4429或MTDH对PC3细胞中MTDH蛋白表达的免疫印迹图;（c）过表达miR-4429或MTDH对PC3细胞迁移的影响;（d）各组迁移细胞数目比较;（e）过表达miR-4429或MTDH对PC3细胞侵袭的影响;（f）各组侵袭细胞数目比较。

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