Effect Of E-cadherin And Alpha SMA Gene Epigenetic Regulation In The Process Of Renal Interstitial Fibrosis Effect

Objective:Pyrosequencing technique was been used for detection of the relationship of E-cadherin and alpha SMA gene promoter region methylation status and gene expression in the EMT process of NRK cells, we can make in-depth understanding of the role of epigenetic in the EMT process of NRK cells, It will help us to further clarify the mechanism of renal interstitial fibrosis, and thus provide a new direction for the prevention and treatment of clinical chronic kidney disease.Methods:Cultured normal NRK cells were divided into two groups: normal control group、TGF-β1intervention group(10ng/ml). After two groups of cells were raised48h, the morphology of cells were observed through inverted phase contrast microscope; the E-cadherin and a-SMA protein expression levels of adherent cells were semi-quantitative detected by the cellular immunocytochemical method; the type I collagen protein content of cells culture supernatant were detected by ELISA; extracted genomic DNA from cells, and the E-cadherin and a-SMA methylation levels of the two groups of cells were respectively detected with the pyrosequencing technology. The Correlation between promoter methylation and gene expression were analysed. Results:1. After48h of cultured, compared with those of normal control, the cell morphology of TGF-β1intervention group changed significantly, many cells showed fibroblast-like morphology changed from the original typical oval shape paving stone like epithelial cell.2. Immunocytochemistry demonstrated that the expression of E-cadherin protein were decrease significantly and the expression of α-SMA protein were obviously increased in TGF-β1intervention group(P<0.001).3. After48h of cultured, compared with those of normal control, the type I collagen content increased significantly in cell culture supernatant of TGF-β1intervention group(P<0.01).4. the E-cadherin and a-SMA gene promoter region methylation of two cell groups were detected by pyrosequencing, the result showed that the promoter region methylation of E-cadherin is higher in TGF-β1intervention group than control group(P<0.05), and the promoter region methylation of α-SMA is lower in TGF-β1intervention group than control group(P<0.05). Conclusion:1. After48h of intervened by TGF-β1(10ng/ml), NRK cells can rapidly establish the ideal model for renal tubular epithelial cell EMT and renal interstitial fibrosis in vitro.2. Which will induced the renal tubular epithelial cells transdifferentiation process that the promoter region CPG island hypermethylation status of E-cadherin gene leads to gene inactivation.3. Which will induced the renal tubular epithelial cells transdifferentiation process that the promoter region CPG island low methylation status of α-SMA gene leads to gene activation.