| Objective: To explore the function and way of exosomes derived from gastric cancer cells,which promoted transdifferentiation of bone marrow mesenchymal stem cells(BMMSC).To further elucidate the molecular mechanism of exosomes in inducing BMMSC transdifferentiation.The present study will provide new experimental basis for gastric cancer microenvironment cell remodeling.Methods: The diameter size and number of exosomes were detected and the amount of exosomes from gastric cancer cell culture supernatant of Rab27a-siRNA interference group were verified by Nanosight.Diameter size and morphology of exosomes were detected by transmission electron microscopy.The protein levels of CD81 and CD9,specific markers of exosomes,were analysed by Western blot.The expression level of ?-SMA in BMMSC before and after induction were detected by immunofluorescence respectively,and the expression of cytokines were detected by qRT-PCR.Transwell migration assay were used to evaluate the migration and invasion ability of gastric cancer cells.The expression of miR-4669 in BMMSC before and after induction was detected and compared by qRT-PCR.Rab27a-si RNA was transfected into SGC-7901 and HGC-27 to inhibit secretory of exosome,and changes of phenotype and function of BMMSC were determinated by immunofluorescence and Transwell migration assay,thus evaluating the induction of exosomes on BMMSC.mi R-4669 mimics was transfected into gastric cancer cells to analyze the changes of phenot ype and function of BMMSC which had been treated with the cell supernatant or exosomes from SGC-7901 and HGC-27 after transfection and to confirm the effect of miR-4669 on transdifferation of BMMSC into gastric cancer tissue-derived mesenchymal stem cells(GC-MSC).The effects of BMMSC in group of mi R-4669 overexpressed on blood vessel formation ability was evaluted by angiogenesis test.The expression changes of some related genes such as CCL2,IL-6,IL-8 in BMMSC,which had been theated with culture supernatant or exosomes from gastric cancer cells transfected with mi R-4669 mimics,were detected by qRT-PCR.The molecular mechanisms of exosomes derived from gastric cancer cells promoting BMMSC to obtain GC-MSC like phenotype was elucidated.Results: Exosomes from gastric cancer cells could reproduce the effect of culture supernatant in inducing BMMSC to obtain GC-MSC like phenotype.Compared with control,induced BMMSC exhibited significantly enhanced expression of ?-SMA and a stronger capacity in promoting migration of gastric cancer cells.Inhibition of exosome secretion by transfecting Rab27a-siRNA into gastric cancer cells,and BMMSC treated with the cell supernatant or exosomes exhibited a weaker phenotype of reactive stroma cells and migration promoting capacity of gastric cancer cells.The expression level of mi R-4669 in BMMSC,which had been induced by exosomes from gastric cancer cells,was significantly increased compared with the control group.When mi R-4669 mimics were transfected into gastric cancer cells,BMMSC,which had been treated by supernatant or exosomes from gastric cancer cells,exhibited a stronger phenotype of reactive stroma cells,migration promoting capacity of gastric cancer cells and a stronger ability to promote angiogenesis.The result of qRT-PCR showed that the expression of pri-4669,pre-4669 and RXRA,target gene of miR-4669,in BMMSC after induction had no significant changes,but the expression of mi R-4669 and inflammatory factors such as CCL2,IL-6 and IL-8 were upregulated.Conclusions: Our findings indicate that exosomes derived from gastric cancer cells is an important component of culture supernatant which induces BMMSC to transform into GC-MSC.Exosomal miR-4669 can be delivered directly to the BMMSC,and then remold them to obtain GC-MSC like phenotype and function.Therefore,miR-4669 is a key signaling molecule in the process of gastric cancer cells paracrine which induces BMMSC transdifferentiation.These findings provide a new mechanism for the remodeling of MSC in microenvironment of gastric cancer. |
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