Down-regulation Of MiR-214 In CAFs Promote Gastric Cancer Cell Migration And Invasion Through Targeting FGF9

Objective Tumor microenvironment is closely related to tumor metastasis.Cancer-associated fibroblasts(CAFs)are one of the most important components of stromal cells in tumor microenvironment.The previous study of our research group found that,micRNA-214(miR-214)expression in CAFs of gastric cancer(GC)was significantly down-regulated,which could play a crucial role in the migration and invasion of gastric cancer cells,but the mechanism was still unclear.In this study,the effect and mechanism of miR-214 in CAFs on the invasion and metastasis of gastric cancer was further discussed,providing new targets and ideas for the clinical treatment of gastric cancer.Methods 1.The CAFs were isolated from human GC tissue and were transfected withmiRNA-214 mimics to upregulate miRNA-214 expression.After that,the effectsof conditioned medium(CM)from CAFs transfected with miRNA-214 mimics onthe proliferation and apoptosis of GC cells were detected by CCK8,Edu and flowcytometry.Immunofluorescence and Western Blot were used to detect theexpression of EMT(epithelial-to-mesenchymal transition)related markers in GCcells,so as to explore whether CAFs-derived miR-214 could promote themigration and invasion of GC cells by activating EMT.2.The effect of miR-214 upregulation in CAFs on the expression of FGF9 wasanalyzed by real-time PCR and Western Blot.Moreover,double luciferasereporter assay was used to verify that FGF9 was the target gene of miR-214.FGF9 neutralizing antibody was added into CAFs-CM to observe the changes ofmigration and invasion ability of gastric cancer cells.3.The expression of miR-214 was detected by in situ hybridization in the tissue ofhuman GC samples,the correlation between the expression of miR-214 and theprognosis was analyzed.Fourthermore,the expression of FGF9 protein wasdetected by immunohistochemistry,and the relationship between the expression ofFGF9 and the clinicopathological characteristics and prognosis of GC wasanalyzed.Results 1.The proliferation and apoptosis capacities of GC cells did not change significantlyafter upregulation of miR-214 in CAFs.However,upregulation of miR-214expression in CAFs significantly increased the expression of E-cad in GC cells,while decreased the expression of Vim,N-cad and Snail,suggesting that miR-214inhibited EMT in GC cells.2.Both the mRNA and protein expression of FGF9 in CAFs were decreased byupregulating miR-214 in CAFs.MiR-214 can directly bind to the 3'UTR region ofFGF9 gene.The migration and invasion abilities of cancer cells were inhibited byadding FGF9 neutralizing antibody to CAFs-CM.3.The expression of miR-214 was detected in the cytoplasm of cancer cells ornormal gastric mucosa epithelial cells,but not in the CAFs and NFs.Moreover,the expression of miR-214 was decreased in cancer cells compared to that innormal gastric mucosa epithelial cells but it did not indicate prognosis.FGF9 ismainly expressed in CAFs,and the expression of CAFs FGF9 in lymph nodemetastases is related to tumor differentiation,Lauren type,and prognosis.Conclusions 1.Upregulation of miR-214 expression in CAFs inhibits EMT of gastric cancer cells,but has no significant effect on proliferation and apoptosis of GC cells.2.FGF9 is a target gene of miR-214 in CAFs,which promotes the migration andinvasion of gastric cancer cells.3.FGF9 is highly expressed in CAFs of gastric cancer tissues,and the expression ofFGF9 in CAFs of lymph node metastases is correlated with tumor differentiation,Lauren type and prognosis.