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. (Chin J Endocrinol Metab, 2016, 32: 685-690) Key words: Keratinocytes; Demethylation; Tumor necrosis factor-α; Matrix metalloproteinase 9
Published Version
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