miR-142-5p通过靶向作用WWP1对前列腺癌细胞PC-3生物学行为的影响

The Effect of miR-142-5p on the Biological Behavior of Prostate Cancer Cell PC-3 by Targeting WWP1

目的探究miR-142-5p与泛素蛋白连接酶1(WWP1)的靶向性及miR-142-5p靶向作用WWP1对前列腺癌细胞PC-3生物学行为的影响。方法采用RT-PCR检测前列腺癌组织和癌旁组织中miR-142-5p与WWP1 mRNA的表达情况;采用荧光素实验验证miR-142-5p与WWP1的靶向关系。体外培养前列腺癌细胞PC-3细胞并分为NC组、miR-142-5p组、WWP1组和miR-142-5p+WWP1组。各组细胞采用对应模拟物转染后,使用Transwell小室实验检测细胞侵袭能力,使用划痕实验检测细胞迁移能力;使用蛋白质印迹实验检测侵袭迁移相关蛋白(E-cadherin,N-cadherin和Vimentin)相对表达水平;采用流式细胞仪检测细胞凋亡率。结果癌组织中miR-142-5p相对表达水平显著低于癌旁组织(0.68±0.16 vs 1.88±0.22;t=19.727;P<0.05);癌组织中WWP1 mRNA相对表达水平显著高于癌旁组织(2.07±0.24 vs 0.24±0.13;t=32.027;P<0.05)。miR-142-5p野生纯合子和WWP1野生纯合子混合组荧光霉素活性显著低于其他各组(P<0.05),荧光霉素验证miR-142-5p和WWP1存在结合位点。相比NC组,miR-142-5p组miR-142-5p相对表达水平显著升高(P<0.05),WWP1水平显著降低(P<0.05);相比NC组,WWP1组miR-142-5p相对表达水平显著降低(P<0.05),WWP1水平显著升高(P<0.05)。相比WWP1组,miR-142-5p+WWP1组miR-142-5p相对表达水平显著升高(P<0.05),WWP1 mRNA相对表达水平显著降低(P<0.05)。相比NC组,miR-142-5p组单位面积内侵袭细胞数目、划痕愈合率、N-cadherin和Vimentin蛋白相对表达水平显著降低(P<0.05),细胞凋亡率和E-cadherin蛋白相对表达水平显著升高(P<0.05);相比NC组,WWP1组单位面积内侵袭细胞数目、划痕愈合率、N-cadherin和Vimentin蛋白相对表达水平显著升高(P<0.05),细胞凋亡率和E-cadherin蛋白相对表达水平显著降低(P<0.05);相比WWP1组,miR-142-5p+WWP1组单位面积内侵袭细胞数目、划痕愈合率、N-cadherin和Vimentin蛋白相对表达水平显著降低(P<0.05),细胞凋亡率和E-cadherin蛋白相对表达水平显著升高(P<0.05)。结论miR-142-5p可以靶向结合WWP1降低前列腺癌组织中WWP1表达,miR-142-5p靶向作用WWP1可抑制体外培养的前列腺癌细胞PC-3的侵袭和迁移,同时促进其凋亡。

Objective To explore the targeting of miR-142-5p and ubiquitin protein ligase 1(WWP1)and the effect of miR-142-5p targeting WWP1 on the biological behavior of prostate cancer cells PC-3.Methods RT-PCR was used to detect the expression of miR-142-5p and WWP1 mRNA in prostate cancer tissues and adjacent tissues.Fluorescein experiment was used to verify the targeting relationship between miR-142-5p and WWP1.Prostate cancer cell PC-3 was cultured in vitro The cells were divided into NC group,miR-142-5p group,WWP1 group and miR-142-5p+WWP1 group.After the cells of each group were transfected with corresponding mimics,the cell invasion ability was detected by Transwell experiment and migration ability was detected by scratch assay.Western blotting was used to detect the relative expression levels of invasion and migration-related proteins(E-cadherin,N-cadherin and Vimentin).Flow cytometry was used to detect the rate of cell apoptosis.Results The relative expression level of miR-142-5p in cancer tissues was significantly lower than that in adjacent tissues(0.68±0.16 vs 1.88±0.22;t=19.727;P<0.05);the relative expression level of WWP1 mRNA in cancer tissues was significantly higher than that in adjacent tissues(2.07±0.24 vs 0.24±0.13;t=32.027;P<0.05).Fluorescein activity in the mixed group of miR-142-5p wild homo-zygote and WWP1 wild homozygous group was significantly lower than that of the other groups(P<0.05).Fluorescein verified that miR-142-5p and WWP1 had binding sites.Compared with the NC group,the relative expression level of miR-142-5p in the miR-142-5p group was significantly increased(P<0.05),and the level of WWP1 was significantly reduced(P<0.05);compared with the NC group,the miR-142-5p in the WWP1 group The relative expression level was significantly reduced(P<0.05),and the level of WWP1 was significantly increased(P<0.05).Compared with the WWP1 group,the relative expression level of miR-142-5p in the miR-142-5p+WWP1 group was significantly increased(P<0.05),and the relative expression level of WWP1 mRNA was significantly reduced(P<0.05).Compared with the NC group,the number of invaded cells per unit area,the scratch healing rate,the relative expression levels of N-cadherin and Vimentin protein in the miR-142-5p group were significantly reduced(P<0.05),and the apoptosis rate and E-cadherin protein expression level was significantly increased(P<0.05);compared with the NC group,the number of invaded cells per unit area,the scratch healing rate,the relative expression levels of N-cadherin and Vimentin proteins in the WWP1 group were significantly increased(P<0.05),and the cell apoptosis and the relative expression level of E-cadherin protein were significantly reduced(P<0.05).Compared with WWP1 group,miR-142-5p+WWP1 group had significantly lower number of invaded cells per unit area,scratch healing rate,relative expression levels of N-cadherin and Vimentin protein(P<0.05),cell apoptosis rate and E-cadherin The relative protein expression level increased significantly(P<0.05).Conclusion miR-142-5p can target WWP1 to reduce the expression of WWP1 in prostate cancer tissues,miR-142-5p targeting WWP1 can inhibit the invasion and migration of prostate cancer cell PC-3 cultured in vitro and promote its apoptosis.