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目的探讨黑色素瘤细胞OCM-1A中表观遗传因素DNA甲基化对HLA-G分子表达的影响。方法利用DNA甲基化抑制剂5-aza-2′-deoxycytidine(ADC)处理HLA-G阴性黑色素瘤细胞OCM-1A,检测经不同浓度、不同时间的ADC处理后HLA-G基因及蛋白表达的变化。序列测定分析ADC处理前后HLA-G基因启动子区220bp片段的甲基化水平,RT-PCR检测HLA-GmRNA水平的变化,Westernblot检测细胞HLA-G分子合成总量以及FACS检测细胞表面HLA-G分子的表达水平。结果HLA-G阴性黑色素瘤细胞OCM-1A经ADC处理后,HLA-G基因启动子区的CpG甲基化程度明显降低,诱导HLA-G基因转录,HLA-G分子获得表达。HLA-G基因及蛋白的表达水平与处理时间相关,随着处理时间的延长,HLA-G表达量上升。结论OCM-1A细胞中,HLA-G基因的表达调控受DNA甲基化影响,ADC对HLA-G的表达调控具有时相性。
Objective To investigate the effect of epigenetic DNA methylation on the expression of HLA-G molecules in melanoma cells OCM-1A. Methods HLA-G negative melanoma cells OCM-1A were treated with DNA methylation inhibitor 5-aza-2’-deoxycytidine (ADC) to detect the expression of HLA-G genes and proteins after different concentrations of ADC treatment Variety. The methylation level of the 220 bp fragment of HLA-G gene promoter region before and after ADC treatment was analyzed by sequencing. The changes of HLA-G mRNA levels were detected by RT-PCR. The total amount of HLA-G molecules synthesized by Western blotting and HLA-G Molecular expression level. Results After treatment of OCM-1A with HLA-G negative melanoma cells, the degree of CpG methylation in the promoter region of HLA-G gene was significantly decreased, and the transcription of HLA-G gene was induced. The expression of HLA-G molecules was obtained. The expression level of HLA-G gene and protein correlated with the treatment time, and the expression of HLA-G increased with the prolongation of treatment time. Conclusions The expression of HLA-G gene in OCM-1A cells is affected by DNA methylation, and the expression of HLA-G in ADC is time-phasic.