| Objective:The overexpression of CDH23 gene was used to study the effects of CDH23 on the morphological transformation,biological behavior of renal clear cell carcinoma cells and tumor formation ability of nude mice.By detecting the effect of CDH23 on MAGI-1 and the expression of EMT-related protein induced by TGF-β1,the molecular mechanism of the effect of CDH23 on the morphological transformation and biological behavior of renal clear cell cancer cells was initially investigated.Method:Human kidney clear cell line Saki-1 was used as the experimental object to stably infect CDH23 overexpression lentivirus vector.RT-qPCR and Western blot were used to detect the overexpression efficiency of CDH23.Two sequences with significant overexpression effect of CDH23 were selected for subsequent experimental study.HE staining and ghost pen cyclic peptide staining were used to detect the effect of CDH23 overexpression on the morphology of renal clear cell carcinoma cells.The effect of overexpression of CDH23 on adhesion of renal clear cell carcinoma cells was detected by cell adhesion assay.The effect of CDH23 overexpression on the proliferation of renal clear cell carcinoma cells was determined by CCK-8,cell cloning and flow cytometry.The effects of CDH23 overexpression on the migration and invasion of renal clear cell carcinoma cells were detected by scratch healing assay and Transwell migration and invasion assay.Tumor formation ability was detected in nude mice.Immunohistochemical staining assays detected the difference in CDH23 expression in human primary and metastatic renal clear cell carcinoma and statistically analyzed.The interaction between MAGI-1 and CDH23 was studied by co-immunoprecipitation and immunofluorescence double staining.The effects of CDH23 overexpression on the expression of EMT-related protein and β-catenin in renal clear cell carcinoma cells were detected by Western blot.HE staining and Western blot were used to detect the morphological changes of CDH23 overexpressed cells and the expression of EMT-related proteins after TGF-β1 induction.Result:A stable caki-1 cell line overexpressing CDH23 was obtained by purinomycin and G418 screening.The results of RT-qPCR and Western blot showed that: Compared with the NC group,the mRNA and protein levels of CDH23 in the experimental group were significantly increased(P<0.01),indicating that a stable overexpression of CDH23 in renal clear cell cancer cells was successfully constructed.The expression of CDH23 was significantly increased in CDH23-Sgr NA11516(P<0.001)and CDH23-Sgrna11514(P<0.01).HE staining showed that the number of spindle cells in renal clear cell carcinoma cells was significantly decreased in the CDH23 overexpression group(P<0.001).Ghost pen cyclic peptide staining showed that the morphology of renal clear cell cancer cells was shortened and rounded,and the cytoskeleton was shortened in the CDH23 overexpression group.Cell adhesion experiment showed that the adhesion ability of renal clear cell cancer cells was significantly enhanced in the CDH23 overexpression group(P<0.0001).CCK-8,plate cloning and flow cytometry showed that the proliferation ability of renal clear cell carcinoma cells in CDH23 overexpression group was not significantly different from that in NC group(ns).Immunohistochemical staining showed that the positive rate of CDH23 in metastatic renal clear cell carcinoma(8/22,36.36%)was significantly lower than that in primary renal clear cell carcinoma(45/60,75%).Meanwhile,statistical analysis of CDH23 expression and clinicopathological features in 60 cases of primary renal clear cell carcinoma showed that The expression of CDH23 in 60 patients with primary renal clear cell carcinoma was correlated with distant metastasis and TNM stage(all p < 0.05),but not with gender,age,tumor diameter or Fuhrman nuclear grade(all p > 0.05).Scratch healing test and Transwell migration test showed that the migration ability of renal clear cell cancer cells in CDH23 overexpression group was significantly decreased(P<0.01).Transwell invasion assay showed that the invasion ability of renal clear cell cancer cells in CDH23 overexpression group was significantly decreased(P<0.01).The CDH23-Sgr NA11516 group with good overexpression effect was selected for tumor formation experiment in nude mice.The experimental results showed that the overexpression of CDH23 had no significant effect on tumor formation ability of nude mice(ns).The results of co-immunoprecipitation and immunofluorescence double staining showed that the interaction between MAGI-1 and CDH23 was found in the CDH23 overexpression group,and the co-localization rate was significantly increased(P<0.01).In the CDH23 overexpression group,EMT-related protein expression was changed,E-cadherin protein expression was increased significantly(P<0.001),and vimentin protein expression was decreased significantly(P<0.01).The expression of β-catenin in renal clear cell carcinoma cells was significantly decreased in CDH23 overexpression group(P<0.001).In the CDH23 overexpression group,the morphology of renal clear cell carcinoma cells was changed and the number of spindle cells was significantly increased after TGF-β1 induction(P<0.01).The expressions of EMTrelated proteins were significantly decreased(P<0.01),while the expressions of vimentin and Snail were significantly increased(P<0.01).Conclusion:Overexpression of CDH23 inhibited the morphological transformation of renal clear cell carcinoma cells,significantly enhanced cell adhesion,and significantly reduced cell migration and invasion.CDH23 could affect the morphological transformation,migration and invasion of renal carcinoma cells.CDH23 plays an inhibitory role in renal carcinoma cells through its interaction with MAGI-1,and TGF-β1 affects EMT process by regulating CDH23 expression.The results of this study provide a theoretical basis for the clinical development of TGF-β1 inhibitors in the treatment of renal cell carcinoma,especially sarcomatoid renal cell carcinoma. |
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