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。
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(DU145,22 rv1,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.
作者
俞莹
李建杰
李汉华
黄娟
翁文浩
陈学飞
YU Ying;LI Jianjie;LI Hanhua;HUANG Juan;WENG Wenhao;CHEN Xuefei(Department of Clinical Laboratory,Yangpu Hospital,Tongji University,Shanghai 200090,China)
出处
《检验医学》
CAS
2021年第12期1267-1273,共7页
Laboratory Medicine
