论文部分内容阅读
目的观察微小RNA-205(miR-205)对前列腺癌细胞PC3迁移能力以及靶基因E盒结合锌指蛋白1(ZEB1)表达的影响。方法采用实时定量PCR检测各前列腺癌细胞株(PC3、LNCa P、DU145)和良性前列腺上皮细胞(Pr EC)中miR-205的mRNA相对表达量,通过miR-205模拟物(miR-205 mimics)、miR-205抑制物(miR-205 inhibitor)和相应的空载体分别转染前列腺癌细胞株PC3后,将PC3细胞分为过表达miR-205组、mimics对照组、抑制miR-205组和inhibitor对照组4组,利用细胞划痕试验和Transwell细胞侵袭试验检测各组细胞迁移和侵袭能力。采用蛋白免疫印迹法检测过表达miR-205组、mimics对照组细胞ZEB1的蛋白表达水平,并通过荧光素酶报告试验验证miR-205与ZEB1基因的直接调控关系。利用蛋白免疫印迹法检测过表达miR-205组、mimics对照组细胞的上皮钙黏素和波形蛋白表达水平。结果前列腺癌细胞DU145、LNCa P和PC3的miR-205 mRNA相对表达量均低于良性前列腺上皮细胞Pr EC(P均<0.01);PC3的miR-205 mRNA相对表达量最低,选为进一步试验的细胞株。24 h后,划痕试验显示过表达miR-205组的迁移细胞数少于mimics对照组,抑制miR-205组的迁移细胞数多于inhibitor对照组(P均<0.01);Transwell细胞侵袭试验显示,过表达miR-205组侵袭细胞数少于mimics对照组,抑制miR-205组侵袭细胞数多于inhibitor对照组(P均<0.01)。过表达miR-205组PC3细胞的ZEB1蛋白表达水平低于mimics对照组(P<0.01)。过表达miR-205组中,携带野生型3’-非翻译区序列载体PC3细胞的荧光素酶活性值低于携带突变型3’-非翻译区序列载体PC3细胞(P<0.01)。与mimics对照组相比,过表达miR-205组的上皮钙黏素蛋白表达水平降低、波形蛋白表达水平升高(P均<0.05)。结论 miR-205可能通过靶向降低ZEB1基因的蛋白表达,抑制前列腺癌细胞的迁移能力。
Objective To investigate the effect of microRNA-205 (miR-205) on the migration of prostate cancer cell PC3 and the expression of target gene E box combined with zinc finger protein 1 (ZEB1). Methods Real-time quantitative PCR was used to detect the relative mRNA expression of miR-205 in prostate cancer cell lines (PC3, LNCa P, DU145) and in benign prostatic epithelial cells (Pr EC). MiR-205 mimics PC3 cells were sub-divided into three groups: miR-205 group, mimics group, miR-205 group and inhibitor of miR-205 In control group, cell migration and invasion were detected by cell scratch assay and Transwell cell invasion assay. The protein expression level of ZEB1 in overexpression miR-205 group and mimics control group was detected by Western blotting, and the direct regulation of miR-205 and ZEB1 gene was verified by luciferase reporter assay. Western blotting was used to detect the expression of E-cadherin and vimentin in overexpression miR-205 group and mimics control group. Results The relative expression levels of miR-205 mRNA in prostate cancer cell DU145, LNCaP and PC3 were lower than those in benign prostatic epithelial cells (all P <0.01). The relative expression level of miR-205 mRNA in PC3 was the lowest Cell lines. After 24 h, scratch test showed that the number of migrated cells in miR-205 group was less than that in mimics control group, the number of migrating cells in miR-205 group was more than that in inhibitor control group (all P <0.01) , And the number of invasion cells in miR-205 group was less than that in mimics control group. The number of invasive cells in miR-205 group was more than that in inhibitor control group (all P <0.01). The expression level of ZEB1 protein in PC3 cells overexpressing miR-205 group was lower than that in mimics control group (P <0.01). In the overexpression miR-205 group, the luciferase activity of PC3 cells carrying the wild-type 3’-untranslated region sequence was lower than that of the PC3 cells carrying the mutant 3’-untranslated region sequence (P <0.01). Compared with the mimics control group, the expression of E-cadherin and overexpression of vimentin in miR-205 group were significantly increased (all P <0.05). Conclusion miR-205 may inhibit the migration of prostate cancer cells by reducing the expression of ZEB1 protein.