SMARCAL1 Regulates The Proliferation Of Embryonic Kidney Cells Through The Wnt Pathway

Objective:To research the influence of the up-regulation of SMARCAL1 gene on the Wnt pathway and the proliferation of embryonic kidney cells.So as to provide experimental basis for researching the pathogenic mechanism of SMARCAL1 gene on kidney disease in Schimke immune-osteodysplasia preliminarilyMethods:We selected an appropriate vector to construct the human SMARCAL1gene over-expression vector,then transfected it into 293T cells for lentivirus packaging,and estimate the level of green fluorescence protein expressing in 293T cells to calculate the lentivirus titer.HEK293 cells were used for the experiment.The experiment was divided into three groups:the blank group without any intervention,negative control group with transfected the unloaded lentivirus and the experimental group with SMARCAL1 gene over-expression.After the transfection of SMARCAL1 over-expressed lentivirus,the transfected cells were screened by observing the green fluorescence protein expression in cells and the purinomycin screening.Verify the stable transfected cells with expression of SMARCAL1 on mRNA level by fluorescence quantitative PCR,and on protein level by Western Blot.CCK-8 and EDU assays were used to detect proliferation of HEK293 cells.The effect of SMARCAL1 gene over-expression on Wnt pathway in HEK293 cells was further investigated by fluorescence quantitative PCR.Results:Plasmid vector sequencing results show that the gene sequence of it is consistent with SMARCAL1 gene.After lentivirus packaging,the titer of SMARCAL1over-expressed lentivirus is 1×10~8TU/m L.The green fluorescence protein in the cells was observed by fluorescence microscopy after 48 hours lentivirus transfection.The results show that the mRNA level of SMARCAL1 in embryonic kidney cells were significantly increased after SMARCAL1 over-expressed lentivirus transfection,as well as the protein level(P<0.05).The lentivirus with SMARCAL1 could be expressed in embryonic kidney cells.CCK-8 and EDU assays both show that the proliferation of cells was decreased(P<0.05),and after SMARCAL1 over-expression,the mRNA levels of CTNNB1 and WNT4 gene of the Wnt pathway in cells were both decreased(P<0.05).It affected the activation of Wnt pathway.Finally to decrease the transcription of Wnt target genes to regulate the cell cycle.Conclusion:In this study,lentivirus vector with SMARCAL1 gene was constructed,and obtained the embryonic kidney cells with SMARCAL1 over-expression successfully.The proliferation of embryonic renal cells HEK-293 was inhibited,and the expressions of CTNNB1 and WNT4 in the Wnt pathway decreased after the up-regulation of SMARCAL1.So we think that SMARCAL1 regulates the proliferation of cells through the Wnt pathway,thereby affecting renal development.