Expression And Significance Of Snail In Renal Tubular Epithelial-mesenchymal Transition

Objective: To investigate the expression of Snail at tissue and cell level,and to analyze the role of Snail in the renal tubular epithelial-mesenchymal transition(EMT)and tubular interstitial fibrosis(TIF).To observe the changes of miRNA expression profile after transfect Snail gene into human renal tubular epithelial cells(HK-2).Methods:(1)Pathological specimens were collected from 40 patients with IgA nephropathy,The pathological changes of renal tubulointerstitium were evaluated by the way of HE,PAS,PASM and Masson staining,then divided into mild,moderate and severe groups according to the degree of renal tubular atrophy and interstitial fibrosis.Four normal kidney tissues were selected as controls.The expressions of Vimentin,SMA,E-cadherin and Snail protein were detected by immunohistochemistry method.(2)The Snail plasmid was constructed and transfected into HK-2 cells with positive liposome LipofectamineTM2000.The morphological changes of cells were observed under inverted microscope after 24 h.(3)The HK-2 cells which cultivated in vitro were divided into blank control group,empty transfection group and Snail gene transfected group.The expression of snail was detected by qPCR,and the expression of snail,Vimentin,?-SMA,E-cadherin were detected by Western blot and RT-PCR in HK-2cells.Furthermore,differentially expressed miRNAs were screened by gene chip.Results:(1)The results of immunohistochemistry showed that Snail,Vimentin and SMA were highly expressed in IgA nephropathy and the expression of E-cadherin was low in IgA nephropathy.Snail was positively correlated with Vimentin and SMA,and negatively correlated with E-cadherin in IgA nephropathy.The higher expression of Snail,the higher degree of TIF.(2)The snail gene was successfully inserted into theplasmid by detecting of plasmid sequences.After transfection for 24 h,the expression of HK-2 cells in the blank control group and the empty transfection group was found to be paving stone.A spindle shaped morphology of HK-2 cells transfected with Snail gene group,the gap widened,there are obvious differences between the two groups.(3)The results of qPCR showed that the relative expression of Snail in Snail gene transfected group was significantly higher than that in the blank control group and the empty transfection group,the difference was statistically significant(P ? 0.01).RT-PCR and Western blot results showed that compared with the control group,the expression of Snail,Vimentin,SMA and Snail in gene and protein levels were increased,E-cadherin protein expression decreased in Snail gene transfected group,the difference was statistically significant(P?0.05).Five distinctly differentially expressed miRNAs were screened by gene chip after transfected Snail gene into HK-2 cells.Then 5026 potential target genes were predicted.Conclusions: The expression of Snail closely with renal tubular epithelial mesenchymal transition and renal interstitial fibrosis;whether differentially expressed miRNAs are involved in Snail promotes the development of EMT and TIF processes to be further studied.