DNA甲基化对miR-133a表达及胶质瘤细胞增殖及凋亡的影响

目的:探讨miR-133a在胶质瘤细胞中的表达及DNA甲基化对其的调控机制并初步探究miR-133a对胶质瘤细胞增殖及凋亡的影响。方法:通过RT-PCR检测胶质瘤组织与正常对照脑组织中miR-133a的表达差异；MSP实验检测胶质瘤组织与正常对照脑组织中miR-133a基因甲基化程度；BSP实验检测正常胶质细胞株HEB与胶质瘤细胞株A172、U87、U251中miR-133a基因的甲基化水平；去甲基化试剂AZA与胶质瘤细胞株A172、U87、U251共培养72 h后，RT-PCR检测上述3株胶质瘤细胞中的miR-133a表达变化；MTT及Annexin V-FITC凋亡实验分别检测AZA处理后的3株胶质瘤细胞的增殖与凋亡能力变化。结果:RT-PCR实验表明与正常对照脑组织相比，胶质瘤组织中miR-133a表达显著下降；MSP实验结果显示，与正常对照脑组织相比，胶质瘤组织中miR-133a基因的甲基化水平明显增高；BSP实验结果显示与正常对照胶质细胞株HEB相比，胶质瘤细胞A172、U87、U251中miR-133a基因的甲基化水平显著增加；RT-PCR实验显示与去甲基化试剂AZA共培养72 h后，胶质瘤细胞株A172、U87、U251细胞中miR-133a的表达显著增加。MTT与Annexin V-FITC凋亡实验结果表明，去甲基化试剂AZA处理后，胶质瘤细胞A172、U87、U251的增殖能力明显下降，细胞的凋亡发生率显著增加。结论:胶质瘤细胞中DNA甲基化通过调控miR-133a的表达而沉默后者发挥抑制肿瘤细胞增殖，促进肿瘤细胞凋亡的功能。

Effect of DNA methylation on the expression of microRNA-133a and the proliferation and apoptosis of glioma cells

Objective:To investigate the expression of microRNA-133a in glioma cells and the regulation mechanism of DNA methylation,and to explore the effect of microRNA-133a on the proliferation and apoptosis of glioma cells.Methods:The expression of microRNA-133a in glioma tissue and normal control brain tissue was detected by RT-PCR.The methylation of microRNA-133a gene in glioma tissue and normal control brain tissue was detected by MSP,and the methylation of microRNA-133a gene in normal glioma cell line HEB and glioma cell lines A172,U87,U251 was detected by BSP.After 72 hours of co-culture of AZA with glioma cell lines A172,U87 and U251,the expression of microRNA-133a was detected by RT-PCR,and the proliferation and apoptotic ability of three glioma cell lines treated with AZA were detected by MTT and Annexin V-FITC respectively.Results:RT-PCR assay showed that the expression of microRNA-133a in glioma tissue was significantly lower than that in normal control brain tissue.MSP assay showed that the methylation level of microRNA-133a gene in glioma tissue was significantly higher than that in normal control brain tissue.BSP assay showed that compared with normal control glioma cell line HEB,glia was significantly higher.The methylation level of microRNA-133a gene in glioma cells A172,U87 and U251 increased significantly.RT-PCR assay showed that the expression of microRNA-133a in glioma cells A172,U87 and U251 increased significantly after 72 hours of co-culture with demethylation reagent AZA.The apoptotic experiments of MTT and Annexin V-FITC showed that the proliferation ability of glioma cells A172,U87 and U251 decreased significantly after treatment with demethylation reagent AZA,and the apoptotic rate of glioma cells increased significantly.Conclusion:DNA methylation in glioma cells silences the expression of microRNA-133a,which can inhibit the proliferation of tumor cells and promote the apoptosis of tumor cells.