地西他滨通过DNA甲基化调控增强结直肠癌细胞中miR-1271-5p的表达和作用  

Decitabine enhances the expression and role of miR-1271-5p in colorectal cancer cells through DNA methylation regulation

作　　者：杨晓莹 刘嘉玉 胡东来 李元[1] 杨华[1] 楚元奎[1,2] YANG Xiaoying;LIU Jiayu;HU Donglai;LI Yuan;YANG Hua;CHU Yuankui(School of Clinical Medicine,Ningxia Medical University,Ningxia Yinchuan 750003,China;Medical Experiment Center,General Hospital,Ningxia Medical University,Ningxia Yinchuan 750003,China)

机构地区：[1]宁夏医科大学临床医学院,宁夏银川750003 [2]宁夏医科大学总医院医学实验中心,宁夏银川750003

出　　处：《中国医院药学杂志》2023年第18期2016-2022,共7页Chinese Journal of Hospital Pharmacy

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

摘　　要：目的:探讨地西他滨(5-Aza-dC,decitabine,DAC)在结直肠癌细胞miR-1271-5p表达调控中的作用和机制。方法:生物信息学分析miR-1271-5p在结直肠腺癌组织和正常结直肠组织中的表达水平,荧光定量-聚合酶链反应(qRT-PCR)检测人结直肠癌细胞系(SW620、HCT116和LOVO)和正常结肠上皮细胞NCM460中miR-1271-5p的表达水平。Western blot验证过表达miR-1271-5p后对HCT116和LOVO细胞增殖和迁移的作用。生物信息学软件预测miR-1271-5p启动子区甲基化岛,甲基化特异性PCR(methylation-specific PCR,MSP)检测miR-1271-5p启动子区的甲基化水平。使用DAC分别处理HCT116和LOVO细胞,MSP和qRT-PCR分别检测DAC对miR-1271-5p启动子甲基化状态和表达的影响。在2种结直肠癌细胞中转染miR-1271-5p抑制物(inhibitor),并同时使用DAC处理细胞,平板克隆实验、Transwell实验和Western blot实验检测细胞的增殖和迁移能力。结果:miR-1271-5p在结直肠腺癌组织和癌细胞中表达均显著下调。miR-1271-5p过表达可显著促进上皮细胞标志物E-cadherin的表达,抑制间质细胞标志物Vimentin及增殖标志物PCNA的表达。miR-1271-5p的启动子区存在多个甲基化岛,且结直肠癌细胞中miR-1271-5p启动子区的甲基化水平显著高于正常结直肠细胞。DAC处理后,HCT116和LOVO细胞中miR-1271-5p启动子区的甲基化水平显著降低,miR-1271-5p的表达显著增强,DNMT3B的表达显著降低。DAC可逆转miR-1271-5p低表达对结直肠癌细胞增殖和迁移的促进作用。结论:抑癌分子miR-1271-5p在结直肠癌细胞中的低表达与其启动子区的高甲基化状态有关,DAC可通过降低miR-1271-5p启动子区的甲基化水平增强其表达,并恢复其抑癌作用。OBJECTIVE To investigate the role and mechanism of decitabine(5-Aza-Dc,DAC)in the regulation of miR-1271-5p expression in colorectal cancer cells.METHODS The expression level of miR-1271-5p in colorectal adenocarcinoma and normal colorectal tissues was analyzed by Bioinformatics.The expression of miR-1271-5p in human colorectal cancer cell lines(SW620,HCT116 and LOVO)and normal colon epithelial cell line NCM460 were detected by fluorescence quantitative PCR(qRT-PCR).The effect of miR-1271-5p overexpression on the proliferation and migration of HCT116 and LOVO cells were verified by Western blot.Bioinformatics software predicts the methylation island of the miR-1271-5p promoter region,and methylation-specific PCR(MSP)detects the methylation level of the miR-1271-5p promoter region.HCT116 and LOVO cells were treated with DAC,and the effects of DAC on the methylation status and expression of miR-1271-5p promoter were detected by MSP and qRT-PCR respectively.The miR-1271-5p inhibitor was transfected into two kinds of colorectal cancer cells,and the cells were treated with DAC at the same time.The proliferation and migration ability of the cells were detected by plate cloning test,Transwelltest and Western blot test.RESULTS The expression of miR-1271-5p was significantly down-regulated in colorectal adenocarcinoma tissues and cancer cells.Overexpression of miR-1271-5p could significantly promote the expression of epithelial cell marker E-cadherin,and inhibit the expression of interstitial cell marker Vimentin and proliferation marker PCNA.There were multiple methylation islands in the promoter region of miR-1271-5p,and the methylation level of the promoter region of miR-1271-5p in colorectal cancer cells was significantly higher than that in colorectal normal cells.After DAC treatment,the methylation level of miR-1271-5p promoter region in HCT116 and LOVO cells was significantly reduced,the expression of miR-1271-5p was significantly increased,and the expression of DNMT3B was significantly decreased.DAC could reverse

关 键 词：miR-1271-5p 结直肠癌 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]