甲基化调控microRNA表达影响胰腺癌细胞增殖、迁移、侵袭的研究

The Study of Methylated Regulation MicroRNA in Pancreatic Carcinoma Cell and Its Effect on Cell Proliferation,Migration and Invasion

目的研究基因启动子区甲基化对胰腺癌相关microRNA(miR34a、miR34b、miR148a、miR203a)表达水平的调控,及其对胰腺癌细胞增殖、迁移、侵袭能力的影响。方法通过5-氮杂-2′-脱氧胞苷(5-Aza-CdR)去甲基化处理胰腺癌细胞MIAPaca-2,使用甲基化特异性PCR(methylation-specific PCR,MSP)法检测60μmol/L5-Aza-CdR处理组及0μmol/L对照组MIAPaca-2细胞目标microRNA启动子甲基化水平的变化;用荧光实时定量PCR检测处理组及对照组目标microRNA表达水平的改变;CCK-8法、划痕实验以及Transwell法检测胰腺癌细胞去甲基化处理后处理组和对照组增殖、迁移、侵袭能力的变化。结果 MSP法结果显示处理组较未处理组目标microRNA甲基化M条带灰度值减弱,非甲基化U条带灰度值增强,处理组目标microRNA启动子甲基化程度较对照组减弱。荧光实时定量PCR结果显示处理组较未处理组目标microRNA相对表达量升高(P<0.05),处理组microRNA的表达水平随去甲基化处理而升高。CCK-8法显示随着药物处理浓度的增加,处理组细胞抑制率增高,去甲基化处理抑制了胰腺癌细胞的增殖能力。划痕实验结果显示处理组较未处理组细胞伤口愈合率降低(P<0.01),去甲基化处理降低了胰腺癌细胞的迁移能力。Transwell法结果显示处理组较未处理组穿膜细胞数减少(P<0.01),去甲基化处理降低了胰腺癌细胞的侵袭能力。结论肿瘤细胞去甲基化处理,导致4种目标microRNA高表达,抑制了胰腺癌细胞的增殖、迁移和侵袭能力。

Objective To study the epigenetic regulation of pancreatic carcinoma related microRNA （miR34a, miR34b, miR148a and miR203a） expression by gene promoter methylation, and its effect on the proliferation, migration and invasion of pancreatic carcinoma cells. Methods The pancreatic carcinoma cells were divided into two groups： control group and treatment group. Control group was treated with 0 μmol/L DNA methyltransferase inhibitor 5-Aza-CdR and treatment group was treated with 60 μmol/L 5-Aza-CdR. The methylation status of microRNA gene promoter regions was detected by MSP （methylation-specific PCR）. The microRNAs’ expression levels were evaluated by real-time PCR. The CCK-8 assay, wound healing assay and Transwell assay were employed to study the proliferation, migration and invasion of pancreatic carcinoma cells, respectively. Results The results of MSP showed that the methylated band of the treated group was weaker than that of the untreated group and the unmethylated band of the treated group was stronger than that of the untreated group. Real-time PCR results showed that the relative expression levels of microRNAs in the treatment group were higher than those in the control group （ P〈0.05）. The CCK-8 assay showed that inhibition rate of the treatment group showed dose-dependent effect with the increase of drug concentration. Wound healing assay showed that the wound healing rate of Treatment group was lower than that of untreated group （ P〈0.01）. The results of transwell assay showed that the number of migrated cells in the treated group was less than that in the untreated group （ P〈0.01）. Conclusion Decreased methylation levels in microRNA promoter region caused by 5-Aza-CdR treatment increased the expression of miR34a, miR34b , miR148a and miR203a, leading to inhibition of the proliferation, migration and invasion of pancreatic carcinoma cells.