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目的:研究CHFR基因启动子甲基化状态与食管癌Eca109细胞增殖和凋亡的关系。方法:分别用不同浓度(2、5、10μmol/L)5-氮杂-2’-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-CdR)处理Eca109细胞,未经药物处理的细胞为对照组。甲基化特异性PCR(methylation-specific PCR,MSP)检测CHRF基因CpG岛的甲基化状态,RT-PCR检测CHFR mRNA的表达情况,MTT法及流式细胞术检测不同浓度5-Aza-CdR处理对Eca109细胞增殖及凋亡的影响。结果:对照组Eca109细胞CHFR CpG岛处于高甲基化状态,5-Aza-CdR处理后CHFR CpG岛可发生不同程度的剂量依赖性的去甲基化。对照组Eca109细胞无CHFR mRNA表达,5-Aza-CdR处理组(2、5、10μmol/L)CHFR mRNA相对表达量显著增加(0.174±0.010、0.221±0.013、0.356±0.014)。不同浓度5-Aza-CdR处理后,Eca109细胞增殖受到抑制[作用72 h时的抑制率分别为(30.87±0.74)%、(44.60±0.79)%、(56.67±0.35)%],细胞凋亡显著增加[(7.46±1.46)%、(16.27±1.61)%、(25.29±2.25)%vs(1.83±0.41)%,P<0.01]。结论:食管癌Eca109细胞经5-Aza-CdR处理后,CHFR基因CpG岛发生部分去甲基化,出现CHFRmRNA表达,并抑制Eca109细胞增殖和促进细胞凋亡。
Objective: To investigate the relationship between the promoter methylation status of CHFR gene and the proliferation and apoptosis of esophageal cancer Eca109 cells. Methods: Eca109 cells were treated with 5-Aza-2’-deoxycytidine (5-Aza-CdR) at different concentrations (2,5,10μmol / L) Of the cells as a control group. The methylation status of CHRF gene CpG island was detected by methylation-specific PCR (MSP). The expression of CHFR mRNA was detected by RT-PCR. MTT assay and flow cytometry were used to detect the methylation status of 5-Aza-CdR Effects of treatment on proliferation and apoptosis of Eca109 cells. Results: The CpG island of CHFR in Eca109 cells in the control group was hypermethylated. There was dose-dependent demethylation of CHFR CpG islands in 5-Aza-CdR treated cells. The expression of CHFR mRNA in 5-Aza-CdR treated group (2, 5, 10μmol / L) increased significantly (0.174 ± 0.010,0.221 ± 0.013,0.356 ± 0.014) in Eca109 cells. After 5-Aza-CdR treatment, the proliferation of Eca109 cells was inhibited [(30.87 ± 0.74)%, (44.60 ± 0.79)%, (56.67 ± 0.35)%] at 72 h, (7.46 ± 1.46)%, (16.27 ± 1.61)%, (25.29 ± 2.25)% vs (1.83 ± 0.41)% respectively, P <0.01]. CONCLUSION: The 5-Aza-CdR treatment of esophageal cancer Eca109 cells partially demethylated the CpG island of CHFR gene, resulting in the expression of CHFR mRNA, inhibiting the proliferation and promoting the apoptosis of Eca109 cells.