Research About The Function Of Demethylation Of Vimentin Gene’s Promoter In Transdifferentiation Of Renal Tubular Epithelial Cells Induced By TGF-β₁

Objective: As the common way to end-stage of chronic kidney disease(CKD) and to complete loss of kidney function, renal interstitial fibrosis (RIF)is regarded as a main link of the process of development disease, and theepithelial to mesenchymal trans-differentiation (epithelial-mesenehymaltransition, EMT) is an important part in the development of RIF, which mainlyperform that the renal interstitial elements are gradually enhancing, such asvimentin, smooth muscle actin (SMA) and collagen, and epithelial componentincreased at the same time. Transforming growth factor beta1(TGF-beta1) isrecognized as one of the main renal fibrosis cell factors, and under certainconditions and TGF-beta1induced, renal tubular epithelial cells can happen theEMT process in vitro. Moreover, DNA methylated modification is an importantmethod of epigenetic modifications, can regulate the methylation status ofcertain gene loci and the gene expression, and cause the corresponding cellphenotypes, and even change the function of cells; As a key enzyme controllinggene methylation’s status, methyltransferase1(DNMT1) may be involved inthe modification of methylation’s process. Therefore, through the observationof cell morphological changes, epithelial cytokeratin (AE1/AE3) and interstitialcytokeratin (vimentin) expression’s changes, DNMT1mRNA, proteinexpression level and the changes of methylation’s status in vimentin’s promoter in the process of trans-differentiation of rat renal tubular epithelial cells (NRK)induced by TGF-beta1in vitro, we aim to investigate the mechanism in theprocess of induced EMT in vitro, and to provide theoretical basis for furtherresearch. Methods: First of all, cultivating rat renal tubular epithelial cells(NRK) in vitro, we divide NRK cells into control group and experimental group(TGF-beta1induced group), and after induced in vitro, we observe the cellmorphological changes with inverted phase contrast microscope, and observethe expression of cytokeratin(AE1/AE3) and vimentin in the two groups byimmunocytochemical method, and then, test the methylation’s status ofvimentin gene’s promoter by the method of bisulfite sequencing PCR, Finally,detect the expression of mRNA and protein of DNMT1using reversetranscription polymerase chain reaction (RT-PCR) and western blot beforeinduced and after. Results: Before and after induced by TGF-β1in vitro, NRKcells’ morphology performs varying degrees of change, and gradually changesto interstitial cell growth form; The grey value measurement and statisticalanalysis show that NRK cells’ DNMT1mRNA and protein expressionsignificantly reduce, and the difference is statistically significant (P<0.05) inthe transdifferentiation model of NRK cells induced by TGF-beta1in vitro; Atthe same time, the methylated status of vimentin’s promoter changes beforeinduced and after, and vimentin’s promoter is to demethylated status afterinduced, also vimentin protein expression increased significantly, and thedifference has statistical significance (P<0.05). Conclusion: After induced byTGF-beta1in vitro, NRK cells happen the transformation from epithelial to mesenchymal cells phenotype, and mainly show to the morphological change ofmesenchymal cells, and also appear epithelial antigenic marker (AE1/AE3) andinterstitial antigenic marker (vimentin) expression increased; the amount ofvimentin expression of NRK cells increases before induced and after may berelated with the changes of methylated status in some vimentin promoter’s geneloci which including the200-300bp sequence in the transcription start site nearthe first exon; the changes of DNMT1mRNA and protein expression beforeinduced and after present that, which may plays a role of regulation in theprocess of changing methylated status of vimentin and may indirectly affect theexpression of vimentin protein, therefore, the methylated status of vimentin’spromoter region and the changes of DNMT1expression may plays an importantrole in the EMT process of NRK cells induced in vitro, and which provides acertain theoretical basis for further research, but the specific mechanismremains to be further elucidated.