5-氮-2′-脱氧胞苷对RKO结肠癌细胞株p16/CDKN2基因去甲基化的转录调节作用  

The study of 5-Aza-2′-deoxycytidine on transcription regulation of p16/CDKN2 gene demethylation in RKO human colorectal cell line

作　　者：方晓明[1] 孙立峰[1] 彭佳萍[1] 董琦[1] 郑树[1] 

机构地区：[1]浙江大学医学院附属第二医院肿瘤研究所,杭州310009

出　　处：《中华医学杂志》2003年第23期2077-2082,共6页National Medical Journal of China

基　　金：国家"九七三"重点基础研究发展规划项目 (G19980 5 12 0 0 );浙江省科技计划项目 ( 0 11110 5 41)

摘　　要：目的　探讨DNA 5′CpG岛去甲基化对RKO结肠癌细胞株p16 CDKN2抑癌基因的转录调控作用及对细胞株生长增殖的生物学影响 ,寻找抗癌治疗的新靶点。方法　用特异性DNA甲基转移酶抑制剂 5 氮 2′ 脱氧胞苷 (5 Aza 2′ deoxycytidine)作用结肠癌细胞株 72h后 ,甲基特异性PCR(Methylation specificPCR ,MSP)、T A克隆及DNA测序分析甲基化状态 ,四唑盐 (MTT)比色、逆转录聚合酶链反应 (RT PCR)、荧光定量PCR(Real timePCR)、免疫组织化学染色 (IHC)及蛋白印迹 (Westernblot)检测用药前后RKO细胞株生长倍增时间以及对p16 CDKN2mRNA及蛋白表达的影响。结果　 (1)未经 5 Aza deoxycytidine作用的对照组RKO细胞基因组DNA的胞嘧啶保持不变 ,而经 5 Aza CdR作用的RKO结肠癌细胞基因组DNA的胞嘧啶均已变为胸腺嘧啶 ;(2 )与对照组相比 ,3组不同浓度 5 Aza 2′ deoxycytidine均能明显抑制肿瘤细胞生长 ,倍增时间分别增加了 1 4 9、1 6 4、1 87倍 ;(3)经 5 Aza 2′ deoxycytidine作用后 ,p16 CDKN2基因mRNA表达分别增加了 4 89、16 91、19 97倍 ,而DNA甲基转移酶mRNA表达显著降低 ;(4 )免疫组化染色和蛋白印迹均显示 ,经处理后的肿瘤细胞p16 CDKN2蛋白表达呈明显增强。结论　DNA异常甲基化与RKO结肠癌细胞有?Objective To explore the transcription regulation of DNA 5′CpG island demethylation on p16/CDKN2 tumor suppressor gene and effects of growth on RKO human colorectal cancer cell line.Methods RKO cell line was exposed to the specific demethylating agent, 5-Aza-2′-deoxycytidine, for seventy-two hours to detect whether the silencing of p16/CDKN2 cell cycle regulatory gene could be reversed.Methylation-specific PCR(MSP), T-A coloning and sequence analysis were evaluated for methylation status.Growth speed, expression of DNA methyltransferase mRNA、 p16/CDKN2 mRNA and protein were determined by MTT assay, reverse transcription polymerase chain reaction(RT-PCR)、 Real-time PCR, immunohistochemistry and western blot. Results (1) All cytosines in CpG dinucleotides in untreated RKO cells with 5-Aza-2′-deoxycytidine remain as C, while all cytosines in treated RKO cells have been converted to thymidine. (2) RKO cell line after treatment with three different concentration 5-Aza-2′-deoxycytidine grew slowly and double time increased to 1.49、1.64、1.87-times respectivly.(3) The expression of RKO cell p16/CDNK2 gene mRNA treated with 5-Aza-2′-deoxycytidine increased to 4.89、16.91、19.97-times respectivly, but the expression of DNA methyltransferanse mRNA was inhibited.(4) Immunohistochemistry and western blot indicated that 5-Aza-2′-deoxycytidine could increase the p16/CDKN2 gene protein expression.Conclusion DNA promoter hypermethylation is asscioated with p16/CDKN2 gene silence in RKO human colorectal cancer cell line.5-Aza-2′-deoxycytidine may effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription silenced by abrreant hypermethylation.

关 键 词：5-氮-2'-脱氧胞苷 结肠癌 p16基因 CDKN2基因 甲基化 细胞增殖 基因表达调控 

分 类 号：R735.3[医药卫生—肿瘤]