启动子区域甲基化状态对THP-1细胞肿瘤坏死因子α分泌的影响

摘要

AIM: To investigate the correlation between the expression and secretion of tumor necrosis factor alpha ( TNF - α ) and the methylation status of TNF - α promoter.METHODS: The secretion level of TNF - α in THP -1 cells stimulated with lipopolysaccharide ( LPS ) for different time was examined by enzyme - linked immunosorbent assay ( ELISA ).The methylation status of TNF - α promoter was measured with bisulfite sequencing analysis.RESULTS: In unstimulated THP - 1 cells, the secretion level of TNF - α was very low, and all the 11 CpG dinucleotides in gene promoter ranging from - 344 bp to transcription start site ( TSS ) were completely methylated.In 4 h LPS - stimulated THP - 1 cells, the secretion level of TNF - α reached peak concentration, while CpG dinucleotides in gene promoter were extensively demethylated and only 4 in 11 were methylated.In 8 h LPS - stimulated THP - 1 cells, the secretion level of TNF - α dropped from the high concentration, and 9 CpG dinucleotides in 11 in gene promoter were methylated.The percentages of methylation between unstimulated THP - 1 cells and 4 h LPS - stimulated THP - 1 cells were statistically different ( P ＜ 0.01 ).The percentages of methylation between 4 h and 8 h LPS - stimulated THP - 1 cells were also statistically different ( P ＜0.05 ).CONCLUSION: The methylation status of TNF - α promoter shows an association with the expression and secretion of TNF - α.%目的:探讨肿瘤坏死因子α(TNF-α)基因启动子区域散在CpG双核苷酸结构甲基化状态对蛋白分泌和基因表达的影响.方法:检测人单核细胞系THP-1细胞在脂多糖(LPS)刺激状态下,不同时点TNF-α的表达和其基因启动子区域的甲基化状态.采用ELISA法测定细胞培养液上清TNF-α水平;采用重亚硫酸盐转化基因测序法测定DNA甲基化状态.结果:受到LPS刺激后,THP-1细胞迅速产生TNF-α,在4 h达到高峰后快速下降.TNF-α基因启动子-344 bp到转录起始位点(TSS)区域内,存在11个散在CpG双核苷酸结构.未受到LPS刺激时均处于甲基化状态;刺激4 h后,有4个处于甲基化状态;刺激8 h后,有9个处于甲基化状态.未刺激与刺激4 h之间,刺激4 h与刺激8 h之间,甲基化率比较均有显著差异(P＜0.01,P<0.05).结论:未受到LPS刺激时,THP-1细胞TNF-α基因启动子区域甲基化水平较高;LPS刺激后,其该区域甲基化水平降低.该变化与TNF-α表达分泌相关.