5-氮杂-2′-脱氧胞苷诱导CDX2基因表达对人胃癌细胞株MKN45增殖和凋亡的影响  

Effect of 5-aza-2'-deoxycytidine Induced CDX2 Gene Expression on Proliferation and Apoptosis of Human Gastric Cancer Cell Line MKN45

作　　者：张健锋[1] 蒋伟[1] 蒯小玲[1] 董丽娟[1] 张弘[2] 鞠少卿[3] 毛振彪[1] 

机构地区：[1]南通大学附属医院消化内科,226000 [2]南通大学附属医院消化病实验室,226000 [3]南通大学附属医院外科综合实验室,226000

出　　处：《胃肠病学》2011年第11期649-653,共5页Chinese Journal of Gastroenterology

基　　金：南通市社会发展基金项目(No;S2009022)资助

摘　　要：背景:DNA甲基化导致基因表达沉默是胃癌发生的重要步骤。近年发现肠表型调节因子CDX2在胃癌中具有抑癌基因的作用。目的:探讨5-氮杂-2'-脱氧胞苷(5-aza-CdR)对人胃癌细胞株MKN45中CDX2基因表达的影响及其对细胞增殖和凋亡的作用。方法:以不同浓度5-aza-CdR(2.5、5、10 Iμmol/L)处理胃癌细胞株MKN45,甲基化特异性PCR(MSP)检测CDX2启动子区甲基化状态;实时荧光定量RT-PCR和蛋白质印迹法分别检测CDX2 mRNA和蛋白表达:CCK8法检测MKN45细胞增殖活性,Annexin V-FITC/PI检测细胞凋亡和Hoechst 33258染色观察其凋亡形态:比色法检测caspase-3活性。结果:MKN45细胞中CDX2基因启动子区高甲基化,CDX2 mRNA表达极低且蛋白不表达:经3种浓度5-aza-CdR处理72 h后,MKN45细胞CDX2基因启动子区高甲基化发生逆转,CDX2 mRNA和蛋白表达均上调。与对照组相比,5-aza-CdR呈剂量依赖性地抑制MKN45细胞增殖和促进凋亡(P<0.05);Hoechst 33258染色示细胞核致密浓染、细胞核碎裂;caspase-3活性随着5-aza-CdR浓度的增加而升高(P<0.05)。结论:MKN45细胞中CDX2基因表达缺失可能与其启动子区CpG岛高甲基化有关;5-aza-CdR能有效逆转CDX2基因的异常甲基化,诱导其再表达.并抑制MKN45细胞增殖和促进凋亡,该作用可能与caspase-3活性有关。Background： Epigenetic gene silencing via DNA methylation is one of the important steps in the development of gastric cancer. CDX2, a regulator of intestinal phenotype, is shown to play a tumor-suppressive role in gastric cancer. Aims： To study the effect of 5-aza-2＇-deoxycytidine （5-aza-CdR） on expression of CDX2 gene in human gastric cancer cell line MKN45 and its action on proliferation and apoptosis. Methods： Gastric cancer cell line MKN45 was treated with different concentrations of 5-aza-CdR （2.5, 5, 10 Ixmol/L）. The methylation status of CDX2 gene promoter region was detected by methylation specific PCR （MSP）. Real-time fluorescent quantitative RT-PCR and Western blotting assay were used to determine the expressions of CDX2 mRNA and protein, respectively. CCK8 assay and Annexin V-FITC/PI were used to examine the proliferative activity and apoptosis, respectively, and the morphology of apoptotic cells was identified by Hoechst 33258 staining. Caspase-3 activity was determined by colorimetric method. Results： Hypermethylation of CDX2 gene promoter region, extremely low expression of CDX2 mRNA and no expression of CDX2 protein were the characters found in MKN45 cells. After treatment with 5-aza-CdR for 72 hours, hypermethylated CDX2 gene promoter region was demethylated, and expressions of CDX2 mRNA and protein were up-regulated in MKN45 cells. Compared with control group, 5-aza-CdR dose-dependently enhanced the inhibition of proliferation and apoptosis of MKN45 cells （P〈0.05）. Hoechst 33258 staining displayed highly condensed chromatin of the nucleus as well as fragments of chromatin. Caspase-3 activity in MKN45 cells was increased in a does-dependently manner after exposure to 5-aza-CdR （P〈0.05）. Conclusions： The CDX2 silencing in MKN45 cells may be related to methylation of 5＇CpG island of CDX2 gene promoter region. 5-aza-CdR may effectively reverse the aberrant methylation of CDX2 gene and induce re-expression of the gene, inhibit the proliferation and enhance apoptos

关 键 词：5-氮杂-2’-脱氧胞苷 CDX2 DNA甲基化 胃肿瘤 细胞增殖 细胞凋亡 CASPASE 3 

分 类 号：R735.2[医药卫生—肿瘤]