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

摘要

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 apoplosis. Methods: Gastric cancer cell line MKN45 was treated with different concentrations of 5-aza-CdR (2.5, 5, 10 μmol/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 apoplosis, 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 demethylaled, 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 MKX45 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 lo methylation of 5'CpG island of CDX2 gene promoter region. 5-aza-CdR may effectively reverse the aberrant methylalion of CDX2 gene and induce re-expression of the gene, inhibit the proliferation and enhance apoptosis of MKN45 cells. This may be related to the activity of caspase-3.%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、10μmol/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.