The Role Of YAP Signaling In Gastric Cancer Derived Mesenchymal Stem Cells In Cancer Progression

Objective: Mesenchymal stem cell(MSC),an important component of the tumor microenvironment,regulate the biological behavior of tumor.In previous research,we have successfully isolated gastric cancer derived-mesenchymal stem cell(GC-MSC)from gastric cancer tissues and found that GC-MSC could promote the development of gastric cancer.However,the role of YAP expression in GC-MSC is unclear.The purpose of this study was to elucidate the role of YAP in GC-MSC in the progression of gastric cancer.Methods: The YAP expression of bone marrow-mesenchymal stem cell(BM-MSC)and GC-MSC were detected by Western blot.The lentivirus YAP-shRNA was used to kockdown YAP in GC-MSC.The efficiency of YAP knockdown in GC-MSC were verified by qRT-PCR and Western blot.Colony formation assay,Western blot,immunofluorescence,cell migration and invasion assays were applied to evaluate growth,migration,invasion,epithelial-mesenchymal transition(EMT)and stemness of GC-MSC.Colony formation assay,immunofluorescence,flow cytometry,qRT-PCR and Western blot were applied to detect the proliferation,apoptosis,migration,invasion and EMT of Blank SGC-7901,GC-MSC(control)-conditioned medium(CM)SGC-7901 and GC-MSC(shYAP)-CM SGC-7901.qRT-PCR was used to detect the expression of angiogenesis associated genes including IL-6,PDGF and VEGF in SGC-7901 with 3 different treatments.Endothelial tube formation assay and migration assay were used to detect the angiogenesis of the CM of SGC-7901 treated with the CM of YAP kockdown GC-MSC.The expression of β-catenin and it’s downstream target gene CD44 and CyclinD in SGC-7901 with 3 different treatments were examined by qRT-PCR and Western blot.In groups of SGC-7901 cells with 3 different treatments after separately inoculation in nude mice tumor model construction,tumor size and weight was determined.Immunofluorescence and immunohistochemistry were used to test the expression of Ki67,β-catenin,E-cadherin,Vimentin and CD31 in tumor tissues.Results: The expression of YAP in GC-MSC was higher than that in BM-MSC.In GC-MSC,YAP knockdown not only inhibited the expression of proliferation associated protein PCNA and Ki67,but also affect the EMT associated protein including decreaseing N-cadherin and Vimentin and increasing E-cadherin.The stemness related proteins including CD44,Sall4 and Nanog also downregulated.Furthermore,the proliferation,migration and invasion of GC-MSC were inhibited.In GC-MSC(shYAP)-CM SGC-7901 cells,the expression of proliferation,migration and stemness related proteins and genes were inhibited.Moreover,the angiogenesis associated genes including IL-8,PDGF and VEGF downregulated.There was no significant change in the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax.The proliferation,migration,invasion and angiogenesis were inhibited.According to the study,the CM of YAP knockdown GC-MSC could inhibit the expression of β-catenin and downstream target genes CD44 and CyclinD.Tumorigenesis in nude mice experiments showed that the growth of subcutaneous xenograft tumor from GC-MSC(shYAP)-CM SGC-7901 cells were inhibited in vivo.The expression of E-cadherin increased,and Ki67,β-catenin,Vimentin and CD31 was obviously decreased in GC-MSC(shYAP)-CM SGC-7901 group mouse tumor tissues.Conclusions: Our findings indicate that YAP knockdown can not only inhibit the profliferation,migration,invasion,EMT and stemness of GC-MSC,but also inhibit the progression of gastric cancer in vitro and vivo.YAP knockdown in GC-MSC may inhibit the gastric cancer progression by regulating β-catenin.