地西他滨通过甲基化调控DU145细胞中miR-3064-5p 的表达  

Decitabine Enhances MiR-3064-5p Expression in DU145 Cells Through Methylation Regulation

作　　者：杨晓莹 胡东来 李元[1] 杨华[1] 楚元奎[1,2] YANG Xiaoying;HU Donglai;LI Yuan;YANG Hua;CHU Yuankui(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;Department of Medical Laboratory,the General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学临床医学院,银川750004 [2]宁夏医科大学总医院医学实验中心,银川750004

出　　处：《宁夏医科大学学报》2023年第4期356-361,369,共7页Journal of Ningxia Medical University

基　　金：国家自然科学基金项目(82160465);宁夏自然科学基金项目(2021AAC03123)。

摘　　要：目的探讨miR-3064-5p在前列腺癌(PCa)细胞中的表达调控机制。方法利用脂质体转染法将阴性对照(NC)组及miR-3064-5p模拟物(miR-3064-5p mimics)转染入PCa细胞DU145中,细胞平板克隆实验、划痕实验、Western blot法分别检测DU145细胞增殖和迁移侵袭能力;qRT-PCR检测前列腺正常上皮细胞及PCa细胞中miR-3064-5p的表达;生物信息学软件分析,甲基化特异性PCR(methylation-specific PCR,MSP)检测miR-3064-5p启动子区的甲基化水平;使用地西他滨(decitabine,DAC)处理DU145细胞,MSP和qRT-PCR分别检测DAC对miR-3064-5p启动子甲基化状态和表达的影响;Western blot检测相关蛋白水平。结果过表达miR-3064-5p可抑制DU145细胞的增殖和转移(P均<0.05);与正常前列腺上皮细胞RWPE-1相比,miR-3064-5p在前列腺癌细胞中表达均下调(P均<0.05);miR-3064-5p的启动子区存在甲基化岛,miR-3064-5p启动子区的甲基化水平高于正常前列腺细胞(P<0.05);DAC处理后,DU145细胞中miR-3064-5p启动子区的甲基化水平降低,甲基化转移酶(DNMT3B)的表达降低,同时,miR-3064-5p的表达增强,并促进了E-Cadherin的表达,抑制了Vimentin及PCNA的表达(P均<0.05)。结论在前列腺癌细胞中DNA甲基化介导了miR-3064-5p的沉默,DAC可降低miR-3064-5p启动子区的甲基化水平,激活其表达,恢复其抑癌作用。Objective To investigate the expression and regulation mechanism of miR-3064-5p in prostate cancer(PCa)cells.Methods The negative control(NC)and miR-3064-5p mimics were transfected into PCa DU145 cells by liposome transfection.The cell clone formation,scratch healing test and Western blot were used to detect the proliferation,migration and invasion of DU145 cells.The expression of miR-3064-5p in normal prostatic epithelial cells and PCa cells was detected by qRT-PCR.Bioinformatics software analysis,methylation level of miR-3064-5p promoter region was detected by methylation-specific PCR(MSP).DU145 cells were treated with decitabine(DAC).The effects of DAC on the methylation status and expression of miR-3064-5p promoter were detected by MSP and qRT-PCR respectively.Western blot was used to detect the levels of related proteins.Results Overexpression of miR-3064-5p significantly inhibited the proliferation and metastasis of DU145 cells(P all<0.05).Compared with normal prostate epithelial cell RWPE-1,miR-3064-5p was significantly down-regulated in prostate cancer cells(P all<0.05).There was a methylation island in the promoter region of miR-3064-5p,and the methylation level of the promoter region of miR-3064-5p was significantly higher than that of normal prostate cells(P<0.05).After DAC treatment,the methylation level of the miR-3064-5p promoter region and the expression of methyltransferase(DNMT3B)in DU145 cells were significantly reduced.At the same time,the expression of miR-3064-5p was increased,which promoted the expression of E-Cadherin and inhibited the expression of Vimentin and PCNA(P all<0.05).Conclusion DNA methylation mediates the silencing of miR-3064-5p in prostate cancer cells.DAC can reduce the methylation level of miR-3064-5p promoter region,activate its expression,and restore its anti-tumor effect.

关 键 词：miR-3064-5p 前列腺癌 地西他滨 DNA甲基化 细胞增殖和迁移 

分 类 号：R737.25[医药卫生—肿瘤]