[High glucose induce the demethylation of CTGF promoter and gene expression]

To investigate the effects of high glucose and 5-aza-2'-deoxycytidine (5-aza-dCyd), a selective DNA methyltransferase inhibitor, on CTGF expression and methylation levels in human glomerular mesangial cells (HMCs).

HMCs were treated with 5-aza-dCyd with different concentrations (0, 1, 2, 5, 10 μmol/L) for 48 h and cell lysates were collected for genomic DNA, total RNA and total protein; HMCs were cultured in DMEM medium containing different concentrations of D-glucose (5 or 30 mmol/L) and cell lysates were collected at different time points (0, 24, 48, 72 h); methylation-specific PCR (MSP) analysis was used to analyze the methylation status of CTGF gene promoter, while RT-PC and western blot were used to evaluate the expression of CTGF mRNA and protein, respectively.

In 5-aza-dCyd treated (48 h) HMCs, CTGF gene promoter was hemi-methylated in 0, 1, 2, or 5 μmol/L groups, and there was low expression of CTGF mRNA and protein with no statistical difference between groups (P>0.05). But its methylation was completely reversed after treated with 10 μmol/L 5-aza-dCyd and the mRNA and protein levels of CTGF in 10 μmol/L group were significantly increased compared to 0 μmol/L group (P<0.01). In the 5 mmol/L D-glucose-treated group, the promoter of CTGF was semi-methylation at all time points, and the expression levels of CTGF mRNA and protein at different time point were all low with no significant difference (P>0.05). However, in the 30 mmol/L group, the semi-methylation of CTGF promoter were detected at 0 h, and it was reduced at 24 h and completely reversed at 48 h. The expression levels of CTGF mRNA and protein at 24, 48, or 72 h in 30 mmol/L group were all significantly elevated compared to 5 mmol/L group(P<0.01).

Both high glucose and 5-aza-dCyd could induce the demethylation process of CTGF gene promoter and increase the expression of CTGF mRNA and protein, indicating that DNA methylation is involved in the regulation of CTGF gene expression in HMCs.