Li Xiaohui, Han Xingtao, Yang Jinhui, Sun Jiantao, Wei Pengtao
Department of Urology, Luoyang Central Hospital, Zhengzhou University, Luoyang 471000, China.
Department of Urology, Luoyang Central Hospital, Zhengzhou University, Luoyang 471000, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Jan;34(1):16-21.
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, 22RV1, 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. MTT 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.
目的 观察微小RNA-519d-3p（miR-519d-3p）对前列腺癌细胞增殖的影响，并探讨其可能的分子机制。方法 采用实时定量PCR检测miR-519d-3p在PC-3、DU-145、22RV1、PC-3M、LNCaP人前列腺癌细胞及RWPE-1人正常前列腺上皮细胞中的表达水平。将miR-519d-3p表达水平最低的前列腺癌细胞转染miR-519d-3p模拟物或阴性对照微小RNA（miR-NC），检测转染效率。采用流式细胞术检测miR-519d-3p对前列腺癌细胞周期的影响。采用MTT法和平板克隆形成试验检测其对前列腺癌细胞增殖的影响。利用生物信息学软件预测并通过双荧光素酶报告基因试验验证miR-519d-3p的靶基因。采用实时定量PCR检测miR-519d-3p靶基因的表达。采用蛋白质免疫印迹分析检测靶基因蛋白及下游蛋白的表达。结果 miR-519d-3p在正常前列腺上皮细胞中的表达明显高于前列腺癌细胞，其中在DU-145细胞中表达最低。转染miR-519d-3p模拟物后，DU-145细胞中miR-519d-3p的表达水平显著升高。生物信息学预测和双荧光素酶报告基因证实肿瘤坏死因子受体相关因子4（TRAF4）是miR-519d-3p的靶基因。miR-519d-3p过表达显著降低前列腺癌细胞中TRAF4基因及其下游TGF-β信号通路蛋白的表达。结论 miR-519d-3p在前列腺癌细胞中表达下调。miR-519d-3p过表达可抑制前列腺癌细胞增殖。其可能机制是miR-519d-3p抑制TRAF4的表达。
