PPP3CB Regulating Cell Growth And Migration Of Renal Cell Carcinoma And The Underlying Mechanism

Background:Renal cell carcinoma(RCC)is a kidney cancer originated from the renal parenchymal urinary tubule epithelial system of malignant tumors.There are more than 209,000 cases and 102,000 deaths worldwide every year.Because the onset of RCC is insidious,about 35% patients suffer from metastasis of RCC at the first time of diagnosis,which subsequently result in bad overall survival.EMT is reported to be a crucial process in tumorigenesis,in which the expression of EMT transcription factors favors migration,invasion,and metastasis.Moreover,PPP3 CB is a subtype of catalytic calcineurin A(CNA)subunit that can make its substrates keep dephosphorylation and PPP3 CB is also reported to regulate cell migration in G401 cells.However,it is unclear whether PPP3 CB plays a critical role in RCC tumorigenesis.Hypothesis:Here,we focused on the PPP3CB-mediated cell biological morphology and tumor growth.Methods:In vitro,we detected cell migrationthrough wound-healing/Transwell assay in 786-O and OSRC-2 cells.In addition,EMT markers and cytoskeleton state were detected by Western-blot/Immunefluorescence and F-actin staining respectively.Moreover,cell proliferation and apoptosis were detected through MTT/CCK-8assay and flow cytometry/pro-apoptotic protein cleaved-Caspase3 respectively in 786-O and OSRC-2 cells.In vivo,tumor formation was carried out in null mice using PPP3CB-overexpressed 786-O cell.Meanwhile,Immunehistochemical(IHC)assay was used to detect PPP3 CB expression in RCC tissue.Results:The results showed that PPP3 CBoverexpression inhibited cell migration,knock-down of PPP3 CB accelerated cell migration compared with the control cell.Fortunately,it was confirmed that PPP3 CB significantly suppressed EMT process and PPP3 CB silence decreased cytoskeleton number and partially destroyed its organization in 786-O and OSRC-2 cells.Specially,PPP3 CB deficiency made the morphology of 786-O cells trend to mesenchyme.Furthermore,we found PPP3 CBinteracted with mesenchymal marker TWIST1 through Co-IP assay and the interaction domain was 1-401 amino acid that is thecatalytic domain of PPP3 CB.Western-blot data indicated that PPP3 CBupregulated TWIST1 protein and its Ser68 phosphorylation.Finally,nude mice tumorigenesis assay showed that PPP3 CBoverexpression inhibited tumor growth.Immunehistochemical result indicated that PPP3 CB expression was higher in para-cancerous tissue compared with RCC tissue,which was consistent with high PPP3CB-associated high survival probability.Conclusion:Together,we defined convinced evidences that PPP3 CBinhibited EMT process and tumor growth through interacting and regulating TWIST1.