The Role And Mechanism Of UBR2 Enriched In P53^-/-m BMMSC-exosome In Gastric Cancer Progression

Objective: Bone marrow mesenchymal stem cell（BMMSC）is an element of tumor microenvironment,however the mechanism of p53 defic ient BMMSC(p53^-/-BMMSC)in tumor microenvironment is not clear.Exosome is the main component of BMMSC paracrine,but the roles and mechanisms of p53^-/-BMMSC-exosome in gastric cancer are unclear.The present study was designed to prove whether UBR2 in p53^-/-BMMSC-exosome could promote the progression of gastric cancer and clarify the molecular mechanism.Methods: The morphology,diameter,and concentration of exosomes derived from p53^+/+mBMMSC and p53^-/-mBMMSC cells were detected by transmission electron microscope and nanopartic le tracking analysis system.The expression levels of exosomal markers CD9 and CD81 were revealed by Western blot.The expression of UBR2 in p53^+/+mBMMSC（mouse bone marrow mesenchymal stem cell,m BMMSC）and p53^-/-mBMMSC cells and exosomes was detected by qRT-PCR and Western blot.CM-Dil-labeled p53^-/-mBMMSC-exosome internalization into p53^+/+m BMMSC cells were determined by confocal microscope and multispectral imaging flow cytometry.Colony formation assay and transwell migration assay were used to analyze the growth ability and migration ability of p53^+/+mBMMSC and MFC cells treated with p53^-/-mBMMSC-exosome.The expression of Nanog,Oct4,Lin28 and EMT（Epithelial-Mesenchymal Transition）associated genes in p53^+/+mBMMSC and MFC cells was detected by q RT-PCR and Western blot.The effects of p53^+/+mBMMSC and MFC cells treated with p53^-/-mBMMSC-exosome on the growth of gastric cancers in vivo were investigated by subcutaneous xenograft tumor model in BALB/c nude mice.QRT-PCR and immunohistochemistry were conducted to detect PCNA,Nanog,Oct4,EMT-associated protein expression and UBR2 in tumor tissues.The concentration of exosomes derived from p53^-/-mBMMSC with UBR2 knockdown were analyzed by nanoparticle tracking analys is system.The expression of Nanog,Oct4,Lin28 and EMT associated genes in p53^-/-mBMMSC with UBR2 knockdown was detected by q RT-PCR and Western blot.Colony formation assay and transwell migration assay were used to analyze the growth ability and migration ability of p53^-/-mBMMSC with UBR2 knockdown.The effects of UBR2-knockdown exosomes on p53^+/+mBMMSC and MFC cells proliferation and migration,and on the expression of UBR2,Nanog,Oct4,Lin28 and EMT associated genes in p53^+/+mBMMSC and MFC cells were detected.Results: Exosomes derived from p53^+/+mBMMSC and p53^-/-mBMMSC displayed classic dish or cup morphology.The concentration of exosomes from p53^-/-mBMMSC was significantly higher than that from p53^+/+mBMMSC.There was no significant difference in the diameter of exosomes from p53^+/+mBMMSC and p53^-/-mBMMSC.All exosomes were expressed exosomal markers CD9 and CD63.P53^-/-mBMMSC-exosome markedly promoted the colony formation and migration of p53^+/+mBMMSC and MFC cells,upregulated Nanog,Oct4 and Lin28 expression,promoted epithelial-mesenchymal transition（EMT）with increased N-cadherin and vimentin expression and decreased E-cadherin expression.P53^+/+mBMMSC and MFC cells treated with p53^-/-mBMMSC-exosome could promote subcutaneous tumor xenograft growth and migration in vivo.UBR2 was highly expressed in p53^-/-m BMMSC cells and exosomes and could be delivered to p53^+/+mBMMSC and MFC cells.UBR2 knockdown in p53^-/-m BMMSC markedly inhibited the colony formation and migration of p53^-/-mBMMSC,downregulated Nanog,Oct4 and Lin28 expression,and inhibited EMT with reduced N-cadherin and vimentin expression and increased E-cadherin expression.Exosome from UBR2-knockdown p53^-/-mBMMSC inhibited the colony formation and migration of p53^+/+mBMMSC and MFC cells markedly,downregulated Nanog,Oct4 and Lin28 expression,and reduced N-cadherin and vimentin expression while increase E-cadherin expression.Downregulation of UBR2 in MFC inhibited the activity of wnt/β-catenin pathway.Conclusions: The concentration of exosomes devired from p53^-/-mBMMSC was significantly higher than that of p53^+/+mBMMSC.Our results indicate that UBR2 in p53^-/-mBMMSC-exosome markedly influence the proliferation and migration of p53^+/+mBMMSC and MFC cells.The exosomal UBR2 may be involved in the progression of gastric cancer via regulating Wnt/β-catenin signaling pathway.