Tumor necrosis factor-αupregulates MMP9 expression through site-specific DNA demethylation
10.3760/cma.j.issn.1000-6699.2016.08.013
- VernacularTitle: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
- Author:
Li LING
;
Meng REN
;
Feng LI
;
Chuan YANG
;
Li YAN
- Publication Type:Journal Article
- Keywords:
Keratinocytes;
Demethylation;
Tumor necrosis factor-α;
Matrix metalloproteinase 9
- From:
Chinese Journal of Endocrinology and Metabolism
2016;32(8):685-690
- CountryChina
- Language:Chinese
-
Abstract:
Objective To determine the involvement of DNA demethylation in tumor necrosis factor-α(TNF-α)-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-α 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.
