| ObjectiveGastric cancer is one of the most common malignant neoplasm which are threatening the health of human.Although surgery is the main method for the treatment of gastric cancer,many patients miss the opportunity of treatment.Therefore,it is critical to explore the metastasis mechanism of gastric cancer and seek effective molecular detection targets which are associated with gastric cancer metastasis.Cancer associated fibroblasts(CAFs),one of the principal constituents of the tumor microenvironment,has closely relation to tumor progression.However,the mechanism is still not clear.Recent studies found that the ecpotic expression of microRNAs(miRNAs)in tumor microenvironment were also associated with various biological behaviors during tumor progression,such as tumor cells proliferation,apoptosis,invasion and metastasis.However,the expression and function of miRNAs in CAFs of gastric cancer is not fully understood.Here we explored the expression level of miRNAs in CAFs and NFs of gastric cancer.Therefor,the aim of this study is to investigate the expression levels of miRNAs in CAFs and NFs of gastric cancer,and to explore the regulation effects of miRNA-214 on FAP(fibroblast activation protein).In addition,this study will further study the effects and mechanism of miRNA-214 on the ability of gastric cancer cells migration and invasion.This study may provide a novel theoretical basis for clarifying metastasis mechanism of gastric cancer and developing new therapeutic target of patients treatment.Methods1.The primary CAFs were isolated from human gastric tumor tissues,while the normal fibroblasts(NFs)were from matched adjacent normal gastric mucosa tissues.The purity of NFs and CAFs is examined through observation of cell morphology,immunocytochemistry staining,transwell assay.2.Real-time PCR was used to detect the expression level of miRNAs in CAFs and NFs.Trough using real-time PCR and western blotting,the expression of FAP at RNA and protein levels were tested when transfected miRNA-214 mimics and negative control into CAFs.3.After transfected miRNA-214 mimics and negative control into CAFs,the transwell assay was used to analyze the effects of miRNA-214 in CAFs on the invasion and migration ability of MGC-803 and SGC-7901.Next,target prediction tools and real-time PCR were used to explore the possible target gene of miRNA-214.Results1.NFs cells were small,and presented with long spindle-shaped;CAFs were spindle and blast-like cells,and they had irregular arrangement.Immunocytochemistry staining showed that primary cultured fibroblast populations(NFs and CAFs)were strongly positive for mesenchymal marker(Vimentin),but were negative for epithelial marker(Cytokeratin).FAP as a special fibroblast biomarker was over-expressed in CAFs when compared to NFs.Real-time PCR and Western blotting assay further confirmed that the RNA and protein expression levels of FAP were significantly increased in CAFs.Transwell assay results revealed that the CAFs could significantly increased the number of migration or invasion gastric cancer cells when compared to NFs.2.Real-time PCR results showed that the expression level of 18 kinds of miRNAs in CAFs was different.Moreover,the expression level of miRNA-214 in CAFs was most observably decreased compared with NFs.After transfecting miRNA-214 mimic into CAFs,the expression of FAP at RNA and protein levels were decreased as compared to the control group.3.Transwell assays results indicated that the conditioned medium from CAFs with high expression of miRNA-214 group could markedly decreased the number of migration or invasion gastric cancer cells when compared to negative group.And increased the expression of miRNA-214 in CAFs significantly decreased FGF9 mRNA levels when compared to control group.This suggested that FGF9 may be a target gene of miRNA-214.Conclusions1.CAFs had significant changes in morphology,immunophenotype and biologicalbehaviors when compared to NFs.2.The expression of miRNAs was different between CAFs and NFs,and the miRNA-214 expression in CAFs was markedly reduced compared with NFs.Over-expression of miRNA-214 in CAFs could inhibit the expression of FAP at RNA and protein levels,this indicated that miRNA-214 might influence the activation of CAFs.3.Over-expression of miRNA-214 in CAFs could decrease the migration and invasion ability of MGC-803 and SGC-7901 cells.miRNA-214 in CAFs might act as a tumor suppressor in gastric cancer progression through inhibiting the expression of FGF9. |
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