过表达miR-519d-3p通过降低DU-145前列腺癌细胞TRAF4水平抑制其增殖

Overexpression of miR-519d-3p inhibits the proliferation of DU-145 prostate cancer cells by reducing TRAF4

目的观察微小RNA-519d-3p(miR-519d-3p)对前列腺癌细胞增殖的影响,并探讨可能的分子机制。方法通过荧光实时定量PCR检测PC-3、DU-145、22RV1、PC-3M、LNCa P人前列腺癌细胞和RWPE-1人正常前列腺上皮细胞中miR-519d-3p的表达水平。以miR-519d-3p表达水平最低的DU-145前列腺癌细胞为转染对象,转染miR-519d-3p模拟物或阴性对照微小RNA(miR-NC)并验证转染效率。采用流式细胞术检测miR-519d-3p对前列腺癌细胞细胞周期的影响,MTT法检测前列腺癌细胞的增殖能力。生物信息学软件预测并应用双荧光素酶报告基因实验验证miR-519d-3p的靶基因。荧光实时定量PCR检测miR-519d-3p靶基因的表达水平,Western blot法检测靶基因蛋白及下游转化生长因子β(TGF-β)信号通路蛋白表达水平。结果与正常前列腺上皮细胞相比,miR-519d-3p在前列腺癌细胞的表达水平显著降低,DU-145细胞表达水平最低。DU-145细胞转染miR-519d-3p模拟物后,细胞miR-519d-3p的表达水平显著增加。过表达miR-519d-3p将前列腺癌细胞的细胞周期阻滞于G0/G1期并降低细胞的增殖能力。生物信息学预测及双荧光素酶报告基因证明肿瘤坏死因子受体相关因子4(TRAF4)为miR-519d-3p的靶基因。过表达miR-519d-3p可明显降低前列腺癌细胞中TRAF4基因及下游TGF-β信号通路蛋白的表达水平。结论 miR-519d-3p在前列腺癌细胞中低表达,过表达miR-519d-3p通过抑制TRAF4的表达抑制DU-145前列腺癌细胞的增殖。

Objective To observe the effect of microRNA-519d-3p （miR-519d-3p） on the proliferation of prostate cancer cells and explore the possible molecular mechanism. Methods The expression level of miR-519d-3p in PC-3, DU-145, 22RVl, PC-3M, LNCaP human prostate cancer cells and RWPE-1 human normal prostate epithelial cells was detected by real-time quantitative PCR. miR-519d-3p mimics or negative control microRNAs （miR-NC） was transfected into the prostate cancer cells with the lowest level of miR-519d-3p expression. Transfection efficiency was examined. The effect of miR-519d-3p on the cell cycle of prostate cancer was detected by flow cytometry. M＇IF assay and plate clone formation assay were used to detect its effect on the proliferation of prostate cancer cells. Bioinformatics software was used to predict and dual luciferase reporter assay was used to validate the target gene of miR-519d-3p. Real-time quantitative PCR was used to detect the expression of miR-519d-3p target gene. Western blot analysis was used to detect the expression of target gene protein and downstream protein. Results The expression of miR-519d-3p in normal prostate epithelial cells was significantly higher than that in prostate cancer cells, and the lowest was found in DU-145 cells. After transfected with miR-519d-3p mimics, the expression level of miR-519d-3p in DU-145 cells increased significantly. Bioinformatics prediction and dual luciferase reporter gene confirmed that tumor necrosis factor receptor associated factor 4 （TRAF4） was the target gene of miR-519d-3p. Overexpression of miR-519d-3p significantly reduced the expression of TRAF4 gene and its downstream TGF-β signaling pathway proteins in the prostate cancer cells. Conclusion The expression of miR-519d-3p is down-regulated in prostate cancer cells. Overexpression of miR-519d-3p can inhibit the proliferation of prostate cancer cells. The possible mechanism is that miR-519d-3p inhibits the expression of TRAF4.