肿瘤坏死因子α诱导位点特异性DNA去甲基化促进基质金属蛋白酶9的表达  

Tumor necrosis factor-a upregulates MMP9 expression through site-specific DNA demethylation

作　　者：凌厉[1] 任萌[2] 黎锋[2] 杨川[2] 严励[2] 

机构地区：[1]广东医学院附属深圳南山医院内分泌科,518052 [2]中山大学孙逸仙纪念医院内分泌科,广州510120J

出　　处：《中华内分泌代谢杂志》2016年第8期685-690,共6页Chinese Journal of Endocrinology and Metabolism

基　　金：国家自然科学基金青年科学基金项目（81400818）

摘　　要：目的研究肿瘤坏死因子α（TNF-α）诱导位点特异性DNA去甲基化促进人表皮角质细胞基质金属蛋白酶9（MMP9）的表达。方法用10ng／ml的TNF-α或2．5μmol／L的5-氮杂-2’-脱氧胞苷（DAC）联合300nmol／L的曲古菌素A（TSA）处理人表皮角质细胞株HaCaT细胞，采用实时荧光定量PCR（Real-time RT-PCR）检测各组细胞MMP9mRNA水平，采用蛋白质印迹（WesternBlot）及酶联免疫吸附法（ELISA）检测MMP9蛋白的表达水平。应用亚硫酸氢钠修饰后测序法（BSP）及甲基化敏感性高分辨率熔解曲线法（Ms-HRM）检测TNF-α干预细胞后MMP9启动子区DNA甲基化改变差异最大的CpG位点。构建不同位点甲基化的MMP9启动子一荧光素酶报告基因重组质粒，检测特异性位点的DNA甲基化状态改变对MMP9启动子转录活性的影响。结果各培养时间点。TNF-α干预组均较相应PBS对照组HaCaT细胞拘MMP9表达量升高，且差异均有统计学意义（P〈0．05）；经实时RT—PCR、蛋白质印迹和ELISA三种方法分别检测到的MMP9mRNA和蛋白表达量随干预时间的变化趋势一致，均为先升高后下降。TNF-α干预后MMP9启动子区的10个CpG位点均发生不同程度的DNA去甲基化改变，其中-36bp位点改变的差异有究计学意义（P〈0．05）。-36bp位点去甲基化后，MMP9转录活性明显提高。TNF-α干预后MMP9启动子爰的10个CpG位点均发生不同程度的DNA去甲基化改变，其中-36bp位点改变的差异有统计学意义：P〈0．05）。-36bp位点去甲基化后，MMP9转录活性明显提高。结论TNF-α通过诱导HaCa]r细胞MMP9基因启动子-36bp位点DNA去甲基化上调MMP9表达。Objective To determine the involvement of DNA demethylation in tumor necrosis factor-ct （ TNF-ct ） -induced matrix metalloproteinase 9 （ MMP9 ） expression in human epidermal keratinocytes. Methods Real-time RT-PCR, Western blot, and enzyme-linked immuno sorbent assay （ ELISA ） were performed to determine the mRNA and protein levels of MMP9 after human keratinocyte cell line （HaCaT） cells were treated with 10 ng/ml TNF-α or 2.5 μmol/L DAC/300 nmol/L TSA. Bisulfite sequencing PCR （BSP） and Methylation-sensitive high- resolution melt analysis （ Ms-HRM ） were used to detect significantly differentially demethylated CpG sites in the human MMP9 promoter region in cells exposed to TNF-α. Different sites methylation constructs of promoter-luciferase reporter gene were made to detect the influences of site-specific DNA demethylation on transcription activity of MMP9 promoter. Results Compared with PBS-treated control, TNF-α significantly increased the expression of MMP9 in HaCaT cells for indicated culture duration （P 〈 0. 05 ）. Real time PCR, Western blot, and ELISA analysis demonstrated that the mRNA and protein levels of MMP9 were increased initially, followed by a decline with prolonged incubation time. After TNF-ct treatment, varied degrees of DNA demethylation occurred at 10 CpG sites in the promoter of MMP9, and the changes at the -36 bp site were statistically significant （ P〈0.05 ）. The demethylation at the -36 bp site greatly increased the transcription activity of MMP9. Conclusion TNF-α promotes MMP9 expression in HaCaT cells through inducing -36 bp site DNA demethylation on the promoter of MMP9.

关 键 词：表皮角质细胞 DNA 去甲基化 肿瘤坏死因子Α 金属基质蛋白酶9 

分 类 号：R587.2[医药卫生—内分泌]