To Study The Effect And Mechanism Of CDH23 Gene Knockdown On The Morphological Transformation And Biological Behavior Of Human Renal Clear Cell Carcinoma Cells

Objective:By studying the effect of CDH23 gene knockdown on the morphology and biological behavior of renal clear cell carcinoma cells,to explore whether CDH23 can promote the sarcoma-like morphological transformation of renal clear cell carcinoma cells and its effects on cell proliferation and invasion;by studying the effects of CDH23 knockdown on cell adhesion,F-actin and cytoskeleton,and detecting the interaction between CDH23 and MAGI-1 and the expression of EMT-related proteins,we preliminarily explored its involvement as an EMT candidate cadherin.Molecular mechanisms of epithelial-mesenchymal transition and renal clear cell carcinosarcoma-like morphological transformation.Method:The human renal clear cell carcinoma cell line 7860 was selected as the experimental object,infected with CDH23 recombinant lentiviral vector,RT-q PCR and Western blot were used to detect the efficiency of CDH23 interference expression,and the two sequences with significantly decreased CDH23 expression were selected for follow-up experimental research,HE staining and phalloidin staining were used to detect the effect of CDH23 knockdown on the morphology of renal clear cell carcinoma cells;CCK-8,plaque cloning and flow cytometry were used to detect CDH23-induced changes in proliferation of clear cell renal cell carcinoma cells.After CDH23 knockdown,the effect of reduced CDH23 expression on migration and invasion of clear cell renal cell carcinoma cells was examined by scratch and transwell cell migration and invasion assays.Cell adhesion,phalloidin staining and cell immunofluorescence double staining experiments were used to explore the changes in the interaction between CDH23 and MAGI-1 in renal clear cell carcinoma cells after CDH23 was down-regulated,E-cadherin and vimentin were measured by the method Western blotting to measure content changes.EMT candidate cadherin,its possible mechanism of involvement in the EMT process and its possible underlying molecular mechanism for promoting renal clear cell carcinosarcoma-like transformation.Result:The lentivirus-infected 7860 cells in each group were observed to have a fluorescence abundance of 80% by fluorescence microscopy.Stable clonal cell lines were obtained by selection of puromycin drugs.The results of both methods,RT-q PCR and Western blot,were suggestive to a comparison with the NC group once,CDH23 expression was significantly reduced in the experimental group at both m RNA and protein levels(P<0.05),it was shown that the kidney cancer cell lines CDH23-sh RNA(1#)with stable CDH23 expression(P<0.05)and CDH23-sh RNA(3#)(P<0.01)were successfully constructed showing the highest interference efficiency;HE staining and phalloidin staining showed that cells in CDH23 knockdown group were spindle-shaped,eosinophilic and fibroblast-like,with elongated cell morphology and obvious atypia.It indicated that the sarcoma-like morphological transformation of renal clear cell carcinoma cells was promoted after CDH23 knockdown.It was found that there was no major change(ns)in proliferative capacity after CDH23 knockdown compared to the NC group as measured by several methods including CCK-8,plate cloning and flow cytometry,the enhanced migration ability of CDH23 knockdown in renal clear cell carcinoma cells was demonstrated by two assays,the scratch healing assay and the Transwell migration assay(P<0.001),CDH23 knockdown in renal clear cell carcinoma cells became more invasive,as detected by the Transwell invasion method(P<0.001);cell adhesion assays were performed to conclude that CDH23 knockdown in renal clear cell carcinoma cells became less adherent(P<0.01);CDH23 knockdown in renal clear cell carcinoma cells had an abnormally elongated cell shape,aggregated F-actin filaments and increased lamellar and filamentous pseudopods,as demonstrated by ghost pencil cyclin staining;cell immunofluorescence double-staining experiments showed that CDH23 knockdown in renal clear cell carcinoma cells co-localized with MAGI-1 at a reduced rate.Significantly decreased(P<0.01);CDH23 knockdown in renal clear cell carcinoma cells showed significant changes in epithelial-mesenchymal transition-related proteins,including decreased expression of E-cadherin protein,and Vimentin protein expression was increased(P<0.01).Conclusion:After the expression of CDH23 is down-regulated,the sarcoma-like morphological transformation of renal clear cell carcinoma cells can be promoted,increased cell migration and invasion ability,significantly reduced cell adhesion,aggregation of F-actin filaments,increased lamellar and filamentous pseudopods,reduced interaction between CDH23 and MAGI-1,and affected EMT-related protein expression.CDH23 was involved in the EMT process of renal clear cell carcinoma cells as a new candidate calmodulin.carcinoma cells,promoting sarcomatoid morphological transformation of renal clear cell carcinoma cells and increasing tumor cell migration and invasive ability.CDH23 may become a new clinical target therapy for clear cell renal cell carcinoma with sarcomatoid differentiation.