Study On The Effect And Epigenetic Mechanisms Of1,25(OH)2D3Regulating Cell Viability Of HaCaT Cells

Objective:Detect the changes of cell viability, globle DNA methylation level, expression and specific methylation status of proliferation and apoptosis related genes in human epidermal keratinocyte cell line HaCaT cells after1,25-dihydroxyvitamin D3(1,25(OH)2D3) treatment to demonstrate the epigenetic mechanisms of vitamin D3treatment in psoriasis preliminarily.Methods:1. HaCaT cells were cultured in DMEM medium with10%fetal serum and treated by10-6M,10-7M and10-8M1,25dihydroxyvitamin D3for24hours after70%confluence of HaCaT cell.2.Cell viability of HaCaT cells treated with different concentrations of1,25(OH)2D3was detected using cell proliferation assay.3. Genomic DNA was extracted using Genomic DNA isolation kit (Tiangen, BeiJing). Total RNA was isolated by Trizol Reagent.4. Globle DNA methylation level was quantified by an ELISA-like reaction using5-methylcytosine antibody.5. mRNA levels of DNA methyl-transferase (DNMTs) and Methyl-Binding Domain Proteins (MBDs) were detected by real-time PCR.6. DNA methylation status of PDCD5and TIMP2in promoter region was detected by methylation-specific PCR (MS-PCR).Results:1. Cell viability of HaCaT cells was decreased after1,25(OH)2D3treatment compared with negative control.2. Globle DNA methylation level was significantly down-regulated in HaCaT cells treated by1,25(OH)2D3compared with negative contol.3. Real-time PCR results showed that DNMT1mRNA levels between the1,25(OH)2D3treatment group and control group have no significant difference. DNMT3a and DNMT3b mRNA levels were significantly decreased (p=0.013). Moreover, MeCP2and MBD2expression was significantly upregulated (p=0.025) and MBD1expression was obviously down-regulated (p=0.014) in the1,25(OH)2D3treatment group compared with control group. No significant difference was observed in MBD3and MBD4expression. 3.PDCD5and TIMP2mRNA levels are significantly increased in the1,25(OH)2D3treatment group compared with negative control (p=0.003, p=0.008). MS-PCR results showed that DNA methylation levels of PDCD5and TIMP2promoter region were significantly decreased in the1,25(OH)2D3treatment group compared with negative control (P=0.013, P=0.037)Conclusion:1,25(OH)2D3can inhibit viability of HaCaT cells and increase expression of PDCD5and TIMP2. DNMT3a and DNMT3b expression was down-regulated by1,25(OH)2D3, which may contribute to the decreased globle DNA methylation level of genomic DNA and specific promoter hypomethylation of PDCD5and TIMP2genes.