5-氮杂-2-脱氧胞苷对MEG3启动子超甲基化逆转及膀胱癌细胞凋亡的作用  

Effects of 5-aza-2-deoxycytidine on reversing hypermethylation of MEG3 gene promoter and apoptosis in bladder cancer cells

作　　者：姜应传 陈炳 邬嘉波 王婕 张小荣 JIANG Yingchuan;CHEN Bing;WU Jiabo;WANG Jie;ZHANG Xiaorong(Department of Urology,Zhoushan Hospital,Zhoushan 316021,China)

机构地区：[1]舟山医院泌尿外科,316021 [2]舟山医院细胞分子生物实验室,316021

出　　处：《浙江医学》2021年第12期1284-1286,1294,I0006,共5页Zhejiang Medical Journal

基　　金：浙江省医药卫生科技计划项目(2017KY680)。

摘　　要：目的探讨5-氮杂-2-脱氧胞苷(5-Aza-CdR)对母系表达基因3(MEG3)启动子超甲基化的逆转以及膀胱癌细胞凋亡的作用。方法以不同浓度的5-Aza-CdR(0、2.5、10μmol/L)作用人膀胱癌T24细胞3 d后,采用甲基化特异性PCR检测MEG3启动子甲基化程度,RT-qPCR法检测MEG3 mRNA表达,Hoechst33528核染色法和流式细胞术检测细胞凋亡情况。结果MEG3启动子甲基化程度随着5-Aza-CdR浓度增加而下降。0、2.5、10μmol/L 5-Aza-CdR作用T24细胞后MEG3 mRNA相对表达量分别为1.00±0.00、3.17±0.14、6.73±0.57,随着5-Aza-CdR浓度增加而增加(均P<0.05)。在荧光显微镜下,0μmol/L 5-Aza-CdR组T24细胞形态基本正常,2.5、10μmol/L 5-Aza-CdR组T24细胞出现不同程度的凋亡。0、2.5、10μmol/L 5-Aza-CdR作用T24细胞后细胞凋亡率分别为(0.34±0.05)%、(11.49±0.36)%、(17.53±2.03)%,随着5-Aza-CdR浓度增加而升高(均P<0.05)。结论5-Aza-CdR可能通过逆转膀胱癌细胞中MEG3启动子超甲基化状态来恢复MEG3 mRNA表达,进而诱发膀胱癌细胞发生凋亡。Objective To investigate the effect of 5-aza-2-deoxycytidine(5-Aza-CdR)on reversing hypermethylation of maternally expressed gene 3(MEG3)gene promoter and apoptosis in bladder cancer cells.Methods Bladder cancer T24 cells were treated with different concentration of 5-Aza-CdR(0,2.5 and 10μmol/L)for 3 days.Then the methylation status of MEG3 promoter were detected by methylation specific PCR(MSP).The expression levels of MEG3 mRNA were detected by real time quantitative RT-PCR(qRT-PCR).Cell apoptosis were examined by Hoechst 3328 nuclear staining and flow cytometry.Results The methylation degree of MEG3 promoter decreased with the increase of 5-Aza-CdR concentration.The relative expression levels of MEG3 mRNA in T24 cells treated with 0,2.5 and 10μmol/L 5-Aza-CdR were 1.00±0.00,3.17±0.14 and 6.73±0.57,respectively(P<0.05).Under the fluorescence microscope,the morphology of T24 cells in the 0μmol/L 5-Aza-CdR group were basically normal,but varying degrees of cell apoptosis were detected in the 2.5 and 10μmol/L 5-Aza-CdR group.The apoptosis rates of T24 cells treated with 0,2.5 and 10μmol/L 5-Aza-CdR were(0.34±0.05)%,(11.49±0.36)%and(17.53±2.03)%,respectively(P<0.05).Conclusion 5-Aza-CdR may restore the expression of MEG3 mRNA and induce apoptosis by reversing the promoter hypermethylation of MEG3 in bladder cancer cells.

关 键 词：膀胱癌 母系表达基因3 甲基化 凋亡 5-氮杂-2-脱氧胞苷 

分 类 号：R737.14[医药卫生—肿瘤]