5-氮-2’-脱氧胞苷对膀胱癌T24细胞株RUNX3表达及生物学行为的影响  

Effects of 5-Aza-2'-deoxycytidine on expression of RUNX3 and biological behaviors of T24 bladder cancer cell line

作　　者：周琦 周祥福[2] 肖运政 张涛[3] 王林[2] 刘志华[2] 

机构地区：[1]西丽人民医院泌尿外科,深圳518055 [2]中山大学附属第三医院泌尿外科 [3]广东省东莞市厚街医院泌尿外科

出　　处：《现代泌尿生殖肿瘤杂志》2009年第4期219-223,共5页Journal of Contemporary Urologic and Reproductive Oncology

摘　　要：目的通过观察DNA启动子区域CpG岛去甲基化对T24膀胱癌细胞株runt相关转录因子3基因(runt-related transcription factor 3 gene,RUNX3)表达及对细胞生物学行为的影响,探讨膀胱癌基因治疗的新靶点。方法甲基化特异性PCR(methylation-specific PCR,MSP)和非甲基化特异性PCR(un-methylation specific PCR,UMP)检测经特异性DNA甲基转移酶抑制剂5-氮-2’-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-CdR)处理前后的膀胱癌T24细胞RUNX3基因的甲基化状态;分别以0.5、5.0和50.0μmol/L5-Aza-CdR处理T24细胞后,半定量RT-PCR检测细胞RUNX3基因mRNA的表达,四唑盐(MTT)比色法观察细胞的增殖活性,流式细胞仪分析细胞的凋亡状况。结果膀胱癌T24细胞RUNX3基因启动子区域CpG岛存在甲基化现象,而经5-Aza-CdR处理后未发现其甲基化;经不同浓度5-Aza-CdR处理后的T24细胞RUNX3mRNA重新表达,且其表达量(0.51±0.06、0.60±0.04、0.69±0.08)与药物剂量呈正相关(F=102.21,P<0.01);与对照组相比,经5-Aza-CdR处理后的T24细胞增殖明显受到抑制,凋亡增加(4.16%±1.56%、25.82%±1.81%、41.77%±3.25%、66.59%±3.16%,P<0.01),且与药物存在剂量依赖关系(F=316.47,P<0.01)。结论T24膀胱癌细胞株RUNX3基因启动子区域CpG岛的甲基化可能是导致该基因表达沉默的主要原因,特异性DNA甲基转移酶抑制剂5-Aza-CdR能够通过逆转T24细胞RUNX3基因的异常甲基化,诱导该基因的重新表达而恢复其抑癌功能。Objective To observe the effect of demethylation of DNA CpG island on the expression of runt-related transcription factor 3 gene（RUNX3） and the growth of T24 bladder cancer cell line,RUNX3 has the potential to become a new target for treatment of bladder cancer. Methods The status of methylation of RUNX3 gene in T24 bladder cancer cell line that was treated with 5- Aza-2＇-deoxycytidine（5-Aza-CdR） was analyzed using methylation specific polymerase chain reaction （MSP） and un-methylation specific polymerase chain reaction（UMP）. After treated with 5-Aza-CdR （0.5,5.0,50.0 μmol/L）, the expression of RUNX3 mRNA was examined by semi-quantitative reverse transcription-polymerase chain reaction（RT-PCR）. Cell proliferation was evaluated by MTT assay,the apoptosis of T24 cells was analyzed by flow cytometry. Results The 5＇CpG island methylation in RUNX3 gene promotor was detected in T24 bladder cancer cell line. After treated with 5- Aza-CdR, the promotor region of RUNX3 gene exhibits demethylation state,and the level of RUNX3 mRNA expression was increased（0.51± 0.06.0.60± 0.04.0. 69±0.08）, and it was significantly correlated with the concentration of 5-Aza-CdR （F=102. 21, P〈0. 01 ）. 5-Aza-CdR obviously decreased the proliferation and increased the apoptosis in T24 cells compared with those in control group（4.16%±1. 56%,25.82%±1.81%,41.77%±3.25%,66.59%±3.16%,P〈0.01）. The inhibition effect of 5-Aza-CdR was dosedependant（F=316.47,P〉0.01）. Conclusions The 5＇CpG island methylation is probably responsible for RUNX3 expression silencing in T24 bladder cancer cell line. 5-Aza-CdR may effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription silenced by aberrant hypermethylation.

关 键 词：膀胱肿瘤 RUNX3基因 5-氮-2'-脱氧胞苷 细胞凋亡 

分 类 号：R737.14[医药卫生—肿瘤]