Effect And Mechanism Of Corin On Preventing The Development Of Mesenchymal Transformation Of Renal Tubular Epithelial Cells By Inhibiting Wnt/β-catenin Signal Pathway

Objective:Previous studies have shown that modulation of the Wnt/β-catenin pathway can reverse the progression of interstitial renal fibrosis.Corin and the Wnt receptor share a similar receptor protein binding site,the CRD structural domain,and pre-experiments have demonstrated an interaction between the two.It is hypothesized that Corin may interfere with the Wnt/β-catenin signalling pathway by binding to Wnt through its CRD structural domain Inhibition of renal fibrosis.Methods:1.Human embryonic kidney cells 293（HEK293）cultured in vitro were used as experimental subjects,and were transfected and divided into double empty plasmid group,Wnt1HA+empty plasmid group,Corin^flag+empty plasmid group,Wnt1^HA+Corin group,Wnt1HA+Corin^ΔCRD-flaggroup,immunoprecipitation was performed and the results of the bands in each group were observed to confirm that Corin can bind to Wnt through the CRD structural domain;2.Human embryonic kidney cells 293（HEK293）were used as the experimental subjects,transfected and divided into the empty group,TGF-β1+Vector group,TGF-β1+Corin group and TGF-β1+Corin^ΔCRD-Flaggroup to observe the cell morphology Western blot analysis of cell lysates:a.Corin and flag expression;b.Detection of activation of Wnt/β-catenin pathway and expression of key downstream target genes regulated by Wnt1,β-catenin,and Snail;c.detection of EMT（Epithelial-Mesenchymal Transition,EMT）and fibrosis-related EMT and fibrosis-related indicators:includingα-SMA,fibronectin and PAI-1 expression;Immunofluorescence detection of fibrosis.3.Statistical analysis was performed using Image J and SPSS26.0 software.Results:1.Successful construction of recombinant plasmids;2.The recombinant plasmids were transfected in HEK293 cells,grouped into blank control group,empty plasmid transfection group,Wnt1+Corin expression co-transfection,Wnt1+Corin with knockdown of CRD structural domain,and immunoprecipitation experiments were performed after successful transfection,and the results suggested that Wnt and Corin could bind,while after knockdown of CRD structural domain,the binding between Wnt and Corin was significantly weakened after knockdown of the CRD structural domain.3.The fibrosis model was constructed using transforming growth factor-β1（transforming growth factor-β1,TGF-β1）,and the groups were divided into control group,TGF-β1+Vector group,TGF-β1+Corin group and TGF-β1+Corin^ΔCRD-flaggroup,and the WB results of protein extraction between the groups showed that,compared with the TGF-β1+Vector group.In the TGF-β1+corin group,β-catenin,α-SMA,Wnt1 and Snail expression levels were significantly lower than in the TGF-β1+null group,while the expression levels of the above proteins were not significantly different in the TGFβ+Corin group with knockdown of the CRD structural domain,suggesting that Corin inhibits the downstream signalling of the Wnt/β-catenin pathway after binding to Wnt through the CRD structural domain.Signalling through the CRD structural domain,inhibiting the pro-fibrotic effect of the Wnt/β-catenin pathway.Conclusions:1.There is an interaction between Corin and Wnt,acting through the CRD structural domain;2.Corin binding to Wnt can affect the Wnt/β-catenin pathway and inhibit renal fibrosis.