The Effect And Mechanism Of Angiogensis In Protein-Protein Interaction Of RI With ANG And Mutant On Bladder Cancer Cells

ObjectiveTo research of angiogenin (ANG) and its mutants with ribonuclease inhibitor (RI) interacting proteins and explore the molecular mechanism of the interaction between mediated and regulated the proliferation of bladder cancer cells.Methods1.PCR mutation reaction construct ANG mutant pCMV-3×flag-ANGH13R and pCMV-3 ×flag-ANGH114R by Pyrobest polymerase and taking pCMV-3×flag-ANG as template. PCR reaction construct pGEX-4T-RI by taking pcDNA3.1-RI as template.2. The interaction of RI and ANG or mutants were detected by CO-IP experiment and Immunofluorescence colocalization in BIU87 cells. GST pull-down showed RI might interact with ANG or its mutants in vitro.3. Through selection of stable cell line pEGFP-Cl-ANGH13R, pEGFP-Cl-ANGH114R (constructed and preserved in our labortary), we found that PI3K/AKT/mTOR pathway related protein had obvious changes, showed that ANG and its mutant could affect tumor progression by this pathway.4. Using the chick embryo chorioallantoic (CAM) assay and tube formation assay,we detected ANG and its mutants the effect of angiogenesis. Application of dual luciferase report assay,we observed the effect of overexpression of RI negatively regulates rRNA transcription.5. Though the experimental animal model of transplantation tumor in BALB/c nude mice,we founed that ANG and mutant can promote the growth of bladder cancer cell.Results1.The expressing plasmid pCMV-3×flag-ANGH13R and pCMV-3×flag-ANGH114Rwas verified by restriction enzyme digestion and Prokaryotic expression vector pGEX-4T-RI constructed correctly.2. RI and ANG or its mutants are present interaction in vivo and in vitro.3. Through selection of stable cell line pEGFP-Cl-ANGH13R, pEGFP-Cl-ANGH1T4R, we showed that in AKT/PI3K/mTOR pathway ANG and mutant could promote relative protein level.4. By The chick embryo chorioallantoic (CAM) vessel formation assay and tube formation assay, ANG and its mutants can promote the formation of tumor blood vessels. By Dual luciferase report gene assay, over expression of RI negatively regulates ANG nuclear activity in enhancing rRNA transcription.5.In BALB/c models, ANG and mutants could promote tumor angiogenesis and the growth of solid tumor.Conclusionribonuclease inhibitor (RI) and angiopoietin (ANG) and its mutants existed interaction in vitro and in vivo. ANG and its mutant can promote tumor angiogenesis. over -xpression of RI negatively regulates ANG nuclear activity in enhancing rRNA transcription.Finally, the interaction between RI and ANG and its mutant protein can regulate the bladder cancer BIU87 cells and influence the growth and tumor vascular proliferation by PI3K/AKT/mTOR signal pathway in bladder cancer.