| Objective:Tumor microenvironment plays an important role in the development and progression of tumor. Non-coding microRNAs (miRNAs) are involved in mediation of microenvironment acting on tumor cells and microenvironment remodeling. Cancer associated mesenchymal stem cells (MSC) as a kind of important microenvironment cells, have been successionally isolated and characterized. However, the origin of cancer associated MSC and the role and mechanism of the aberrant expression of miRNAs in their activation are still unclear. Bone marrow derived MSC (BM-MSC) are important precursors of microenvironment cells. Studies have uncovered that gastric cancer tissue derived MSC (GC-MSC) exhibit similar superficial markers and mulitpotency to BM-MSC, but have stronger capacity of proliferation and promoting gastric cancer. The present study aims to establish the trans-differentiational model of gastric cancer inducing BM-MSC, screen for differential expression of miRNAs in GC-MSC, take the miRNAs as an entry point, identify key miRNAs regulating BM-MSC differentiation into GC-MSC and clarify the mechanism of miRNA reprogramming BM-MSC.Methods:GC-MSC, adjacent non-cancerous gastric tissue derived MSC, human BM-MSC and 615 murine BM-MSC were isolated and cultured. Gastric cancer cell conditioned medium were used to treat BM-MSC and establish trans-differentiational model of BM-MSC in vitro. Agilent human miRNA array were used to screen for differential expression of miRNAs between GC-MSC and adjacent non-cancerous MSC. Further the differential miRNAs were tested by quantitative RT-PCR in GC-MSC and gastric cancer inducing BM-MSC, which were used to confirm the differential expression of miRNAs. BM-MSC were transfected with miRNAs inhibitor to suppress the miRNA expression and mimic the aberrant expression of miRNAs in GC-MSC. a-SMA and FAP expression were tested by immunofluorescent staining. The content of cytokines in cell culture medium were determined by luminex. These were used for phenotype analysis. Cell culture medium were collected from BM-MSC and treated gastric cancer cells. Colony formation assay, transwell migration and invasion assay, and tumorigenesis in nude mice were performed to analyze the function of transfected BM-MSC ingastric cancer. GC-MSC were transfected with miRNA mimics to over-express miRNAs. Then the phenotype and function of GC-MSC were tested to evaluated the role of miRNAs in retro-differentiation of GC-MSC. Target prediction,3'UTR reporter vector construction and western blot were used to detect and determine target of miRNAs. Based on this, the targets were interfered in gastric cancer inducing BM-MSC, miRNAs transfected BM-MSC, GC-MSC and GC-MSC, then the phenotype, function and related signaling pathway activation in MSC were analyzed to evaluated the role of targets in tans-differentiation.Results:The results of miRNA array showed that there are 61 miRNAs with fold change>2 and P< 0.01, which included 38 higer expression and 23 lower expression of miRNAs. These differential miRNAs associated with tumor regulation were randomly selected and tested in 5 pairs of GC-MSC and adjacent non-cancerous MSC. The results showed that miR-99a-5p expression level in GC-MSC were significantly lowere than the adjacent non-cancerous MSC. The cell model of gastric cancer conditioned medium inducing trans-differentiation of BM-MSC to GC-MSC-like cells were successfully established. Quantitative RT-PCR analysis revealed that miR-99a-5p were obviously reduced in induced BM-MSC. Inhibiton of miR-99a-5p in BM-MSC could remarkedly promote a-SMA and FAP expression. Luminex assay showed that the content of IL-6, IL-8, MCP-1, RANTES and so on were increased in transfected BM-MSC. The conditioned medium collected from the transfected BM-MSC were used to treat gastric cancer cells and promoted the ability of colony formation, migration, invasion and tumorigenesis of gastric cancer cells. Overexpression of miR-99a-5p blocked gastric cancer conditioned medium inducing BM-MSC. Upregulatio of miR-99a-5p in GC-MSC significantly inhibited a-SMA and FAP expression, cytokines secretion, block their role in promoting gastric cancer cell proliferation, migration and invasion. Target prediction and luciferase reporter assay showed that FGFR3 and IGF1R were targets of miR-99a-5p. Western blot analysis revealed that the protein leves of FGFR3 and IGF1R and their downstream signal moleculars AKT and ERK were obviously increased and activated in gastric cancer conditioned medium inducing BM-MSC, miR-99a-5p inhibitor transfected BM-MSC and GC-MSC. FGFR3 inhibitor AZD4547 and IGF1R inhibitor OSI-906 were used to treat BM-MSC, respectively. The data indicated that AZD4547 treatment could inhibit gastric cancer cell conditioned medium or miRNA inhibitor promoting a-SMA and FAP expression and activation of downstream signaling pathway, and block BM-MSC prompt gastric cancer cells. AZD4547 treatment also directly inhibited a-SMA and FAP expression in GC-MSC and abolished their supporting role in gastric cancer cells. While OSI-906 pretreatment did not affect the role of gastric cancer cell conditioned medium or miRNA inhibitor in trans-differentiation of BM-MSC.Conclusion:The model of gastric cancer cell conditioned medium inducing trans-differentiation of BM-MSC into GC-MSC in vitro was successfully established. MiR-99a-5p were found to be downregulated in GC-MSC. Knock down of miR-99a-5p induced trans-differentiation of BM-MSC into GC-MSC, while overexpression of miR-99a-5p promoted retro-differentiation of GC-MSC into BM-MSC. FGFR3 was the key molecular mediating gastric cancer cell conditioned medium and miR-99a-5p reprogramming BM-MSC. The finish of the study will clarify the role and mechanism of miRNAs regulating trans-differentiation of BM-MSC into GC-MSC, provide new experimental evidence for the derivin and remodeling of gastric cancer microenvironment cell GC-MSC and establish new targets aiming at microenvironment and therapeutic strategy, which have important scientific investigation significance and clinical potential. |
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