The Role And Mechanisms Of Gastric Cancer Tissue Derived Mesenchymal Stem Cells Promoting Gastric Cancer Cells Metastasis By Upregulating MiR-4669

Objective: To explore the role of miR-4669 mediating gastric cancer tissue derived mesenchymal stem cells(GC-MSC)promoting gastric cancer metastasis and elucidate its molecular mechanism.Methods: Transwell migration assay,Western blot and qRT-PCR were used to detect the migration capacity and the expression levels of Sall4,EMT protein markers and miR-4669 in gastric cancer cells after treated with GC-MSC conditioned medium in vitro.In vivo experiment,BALB/c nude mice were injected intraperitoneally with gastric cancer cells to detect the peritoneal metastasis ability of gastric cancer cells.mi R-4669 mimics was used to transfect gastric cancer cells,and the metastasis ability of gastric cancer cells was observed before and after transfection.Gastric cancer cells pre-transfected with mi R-4669 inhibitor were then treated with GC-MSC conditioned medium to observe the effect of miR-4669 on the metastasis ability of gastric cancer cells.TargetScan and PicTar were used to predict the target gene of miR-4669.qRT-PCR,Western blot and luciferase reporter assays were used to detect and validate the potential target gene of miR-4669.RNA interference or gene overexpression vector co-transfected with miR-4669 mimics or co-treated with GC-MSC conditioned medium were used to treat gastric cancer cells,which were used to analyze the role of target gene in the process of GC-MSC and miR-4669 promoting gastric cancer metastasis.Results: GC-MSC conditioned medium significantly promoted the metastasis of gastric cancer cells in vitro and in vivo,upregulated the mRNA and protein expression levels of Sall4,suppressed the expression levels of E-cadherin,induced vimentin expression and increased the level of miR-4669 in gastric cancer cells.The transfection of mi R-4669 mimics to gastric cancer cells significantly up-regulated the expression levels of mi R-4669 and promoted the metastasis of gastric cancer cells in vitro and in vivo.It could also enhance the mRNA and protein expression of Sall4 and induce EMT.The effect of miR-4669 overexpression on gastric cancer cells was similar to that of GC-MSC conditioned medium.Gastric cancer cells pre-transfected with mi R-4669 inhibitor could block the effect of GC-MSC conditioned medium on gastric cancer cells.Target gene prediction,luciferase reporter assay,q RT-PCR and Western blot detection confirmed that TIMP3 was the target gene of miR-4669.Knockdown of TIMP3 in gastric cancer cells by RNA interference could recapture the function of miR-4669 mimics and GC-MSC conditioned medium promoting gastric cancer cells metastasis.Conversely,TIMP3 overexpression abolished the metastasis-promoting function of miR-4669 mimics and GC-MSC conditioned medium in gastric cancer cells.Conclusions: GC-MSC promotes gastric cancer cells metastasis by upregulation of miR-4669 and suppression of TIMP3.This study will clarify a new molecular mechanism of gastric cancer microenvironmental cells GC-MSC promoting gastric cancer development and provide an effective therapeutic target for the treatment of gastric cancer.