The Effect Of Protein-protein Interaction Of Ri With Ang On Bladder Cancer Cells

ObjectiveTo study the interaction between RI and ANG, further the effect and themechanism on the growth on bladder cancer cells.Methods1. With pCMV-3×flag-ANG as a template, PCR amplified mutantobtained ANG pCMV-3×flag-ANGH37A. With pcDNA3.1-RI as a template,PCR amplified RI coding sequence, cloned into pGEX-4T-1prokaryoticexpression vector, prokaryotic expression vector pGEX-4T-RI. Then wereto pCMV-3×flag-ANG, pCMV-3×flag-ANGH37Aand pcDNA3.1-RI as atemplate, PCR amplification of the coding region sequence methodeukaryotic expression plasmid pEYFP-ANG, pEYFP-ANGH37Aand pECFP-RI.2. RI and ANG or its mutants conducted in HEK293cells and T24cells with co-immunoprecipitation experiments and fluorescence resonanceenergy transfer experiments. The studies showed RI can interact with ANGor its mutants in cells. In vitro studies, GST pull-down showed RI might interact with ANG or its mutants in vitro. Likewise, RI co-localized withANG and its mutants in T24cells by immunofluorescence experiments.3. The pCMV-3×flag-ANG and pCMV-3×flag-ANGH37AtransfectedT24cells detect effects on T24cells and the signaling pathway proteins forPI3K/AKT/mTOR impact.4. Xenograft model to builded, test for tumor growth and angiogenesis,for the impact of ANG protein and its mutants.Results1. A recombinant eukaryotic expression plasmid pCMV-3×flag-ANGH37A,pEYFP-ANG, pEYFP-ANGH37Aand pECFP-RI constructedcorrectly. Prokaryotic expression vector pGEX-4T-RI constructed correctly.2. In vivo and in vitro, RI and its mutants are present ANGinteraction.3. After passing pCMV-3×flag-ANG and pCMV-3×flag-ANGH37Atransfected T24cells, which promote the growth of T24throughPI3K/AKT/mTOR pathway.4. In animal models, ANG protein and its mutants to promote thegrowth of tumors and increased neovascularization.ConclusionThe eukaryotic expression plasmid pCMV-3×flag-ANGH37A,pEYFP-ANG, pEYFP-ANGH37A, pECFP-RI and prokaryotic expression vector pGEX-4T-RI. RI and ANG or its mutant were proved the presence ofprotein interactions. RI could interact with ANG or its mutants, thusaffecting the growth of bladder cancer T24in PI3K/AKT/mTOR signalpathway.