Analysis Of Pax2Gene DNA Methylation Status In Primary Vesicoureteral Reflux

BackgroundPrimary vesicoureteral reflux (VUR) is one of the most common congenital anomalies of the kidney and urinary tract, and can lead to renal failure. VUR has a genetic basis, the incidence of VUR in compatriots are significantly higher than in healthy children. Animal model studies found a series of genes involved in the development of the urinary system had relationship with VUR. But the genome-wide scan in primary VUR family or sporadic patients had not found these candidate genes associated with primary VUR. So the exact pathogenesis of primary VUR remains unclear.Pax2gene encodes a protein of nuclear transcription factor. In the development of urinary system, Pax2express in pronephron, mesonephric duct, metanephros and mesenchymal cells and has three major roles, including the coordination of sprouting and positioning of ureteric bud, inhibition of the apoptosis of ureteric bud and promoting the conversion of mesenchymal cells to epithelial cells.Our preliminary studies found Pax2protein abundantly expressed in VUR ureteral epithelial cells but showed a trace expression in normal ureteral epithelial cells. Research work also found the decreased expression of Pax2in the development of mouse kidney was accompanied with the increase of Pax2DNA methylation level. So we assume that the expression of Pax2in VUR ureteral tissue may associated with the change of Pax2DNA methylation. On the basis of the above understanding, we first detect the expression of Pax2mRNA and protein in VUR and control group, and then use bisulfite sequencing PCR (BSP) to detect Pax2DNA methylation status. Explore possible molecular mechanism of VUR from the epigenetic point of view. Objective (1) To detect the expression of Pax2mRNA and protein in human ureteral tissue.(2) To detect Pax2gene DNA methylation status.(3) To analyze the possible causes of the differences of the DNA methylation status by detecting the mRNA of DNA methyltransferases.Method Immunohistochemistry, western blot and real-time PCR were used to detect the expression of Pax2protein and mRNA, and then BSP were used to detect Pax2gene DNA methylation level, and finally real-time PCR was used to detect DNA methyltransferase in ureter tissue between VUR and control groups.Result (1) The results showed that Pax2expressed in the nuclei of ureter epithelial cell in VUR, and the expression of protein (P=0.011) and mRNA (P<0.0001) in VUR group were significantly higher than in control group.(2) There was no significant difference in the expression of Pax2in different age (P=0.309), gender (P=0.316) and reflux grade (P=0.339) of VUR.(3) BSP showed Pax2gene DNA promoter methylation level in VUR group was significantly lower than that in control group (P=0.045), but there was no significant correlation with Pax2mRNA expression (P=0.091).(4) There was no significant difference of Pax2DNA methylation in different age (P=0.574), gender (P-0.347) and reflux grade (P=0.881) of VUR.(5) The expression of Dnmt3a mRNA in VUR group was significantly lower than in control group (P=0.027).Conclusion(1)The expression of Pax2gene in VUR was significantly higher than in control group and Pax2gene DNA methylation was participate in the regulation of Pax2expression (2) The hypomethylation of Pax2gene DNA methylation may be due to the decrease of Dnmt3a.