人胃癌细胞肿瘤相关基因的表达与甲基化调控  

Expression and methylation of tumor-associated genes in human gastric cancer cell lines

作　　者：杨丽[1] 朱红音[2] 程中华[2] 陆嵘[2] 陈萦晅[2] 房静远[2] 

机构地区：[1]同济大学附属同济医院消化科,上海市200065 [2]上海第二医科大学附属仁济医院消化疾病研究所,上海市200001

出　　处：《世界华人消化杂志》2005年第13期1493-1498,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30170413~~

摘　　要：目的:旨在阐明胃癌的发生中多种抑癌基因和癌基因的甲基化情况,以期为进一步深入探索通过改变抑癌基因的甲基化而为胃癌治疗提供新方法. 方法:培养人胃癌细胞株MKN-45和HGC-27两种细胞,在MTT确定去甲基化制剂5-氮脱氧胞苷(5-aza- 2’-deoxycytidine,5-aza-dC)浓度和时间对细胞生长活力没有影响后,分别以2,5,和10μmol/L,浓度分别干预24和72 h,然后提取其DNA和RNA,用RT-PCR的方法检测p16INK4A,p21WAF1,p53,c-Ha-ras和c- myc等多种基因的表达情况;同时以流式细胞仪分析药物干预后的MKN-45和HGC-27的细胞周期变化; DNA分析则通过亚硫酸氢盐修饰和测序和甲基化特异性PCR(MSP)的方法检测p16INK4A基因及c-myc启动子区甲基化的情况. 结果:我们所采用的5-aza-dC的浓度和时间对细胞的生长无显著性影响.5-aza-dC干预前,MKN-45和HGC-27两种胃癌细胞系中均有p16INK4A表达,5-aza- dC干预后在MKN-45和HGC-27两种胃癌细胞系中p16INK4A的表达增强,且不同的胃癌细胞株表达增强最明显时的时间与浓度不同.-p53,p21WAF1,c-myc,c-Ha-ras 等多种基因在干预前后均有表达,且在干预前后无明显变化.在5-aza-dC干预后,HGC-27细胞周期阻滞在G1期,而MKN-45细胞的周期无明显改变.p16INK4A启动子区存在甲基化使得该基因的表达减少,在去甲基化试剂处理后使其表达增强. 结论:人胃癌细胞系MKN-45和HGC-27中,p16INK4A 启动子甲基化是其在表达减弱的主要原因,其去甲基化程度取决于5-aza-dC干预的时间和浓度.AIM: To investigate the expression and methylation of tumor suppressor genes and oncogenes in the carcinogen-esis of gastric cancer, and to further explore new methods for the treatment of gastric cancer. METHODS: The gastric cancer MKN-45 and HGC-27 cell lines were cultured and then exposed to different concentrations (2 μmol/L, 5 μmol/L and 10 μmol/L) of 5-aza-2'-deoxycytidine (5-aza-dC) for 24 and 72 hours. MTT assay was used to examine the viability of the cells. Then the DMA and RNA of the cells were extracted and the expression of p16INK4A, p21WAF1, p53, c-myc, and c-Ha-ras were detected by reverse transcription polymerase chain reaction (RT-PCR). At the same time, the cell cycles of MKN-45 and HGC-27 were observed by flow cytometry. Bisulfite modification and sequencing and methylation-specific PCR were used to detect the methylation of p16INK4A and c-myc promoter region. RESULTS: The concentrations and exposed time of 5-aza-dC had no significant effect on the viability of gastric cancer cells. p16INK4A was expressed in both MKN-45 and HGC-27 cells before treatment. After treated with 5-aza-dC, p16INK4A expression was increased in both kinds of the cells, and the 5-aza-dC concentration and exposed time were different between the two kinds of cells when the most markedly increased expression of p16INK4A appeared. p53, p21WAF1, c-myc and c-Ha-ras were all expressed before and after treatment. HGC-27 cells were blocked at G1 period, but no changes of MKN-45 cell cycle were observed. Methylation in p16INK4A promoter region occurred so that the expression of this gene was reduced. After treated with demethylation agent 5-aza-dC, the expression of p16INK4A was increased. CONCLUSION: Methylation regulates the expression of p16INK4A,but not p21WAF1,p53,c-myc,and c-Ha-ras. 5- aza-dC can up-regulate the transcription of tumor suppressor gene through demethylation, in which its concentration and exposed time play an important role.

关 键 词：肿瘤相关基因 5-AZA-DC P16^INK4A基因 P21^WAF1 C-HA-RAS HGC-27 癌细胞株MKN-45 P16^INK4A MKN-45细胞 c-myc 5-氮脱氧胞苷 启动子区甲基化 流式细胞仪分析 调控 去甲基化制剂 细胞生长活力 RT-PCR 甲基化特异性 表达增强 

分 类 号：R735.2[医药卫生—肿瘤]