The Role And Mechanism Of MiR-3189-3p And CFL2 Gene In Gastric Cancer Cells MGC803

Introduction: Gastric cancer(GC)is one of the most frequent malignancies of the digestive system,the outcome of the current clinical treatment is still not satisfactory.So,elucidating the molecular mechanism of the development and progression of gastric cancer has important implications for developing novel therapeutic targets and improving the prognosis of patients with gastric cancer.S100A4 is a member of the S100 family of calcium binding proteins.Our previous studies have demonstrated that high expression of S100A4 is closely related to proliferation,migration and apoptosis of GC cells.GDF15 is an important downstream gene of S100A4 and is involved in mediating the effects of S100A4 on proliferation,apoptosis and CSC-like properties of GC cells.MiR-3189-3p lies in intron of GDF15.Currently,studies have shown that miR-3189-3p exerts different biological functions in different types of tumor,but the expression status and function of miR-3189-3p in GC cells remains unknown.Given that S100A4 can regulate GDF15 expression,we speculated that S100A4 may also be involved in the regulation of mi R-3189-3p expression,and miR-3189-3p may affect the biological function of S100A4.The target genes downstream of miR-3189-3p were predicted by bioinformatic software,and CFL2(cofilin-2)was selected as a candidate target gene according to the relevant expression and function.CFL2 is a member of the ADF/cofilins family of actin-binding proteins.Previous studies found that CFL2 is mainly expressed in muscle tissue and participates in muscle development and maintenance.Recent studies have found that CFL2 is expressed in a variety of tumor tissues such as breast cancer,glioblastoma and pancreatic cancer and exerts different biological functions.But so far,the role of CFL2 in GC cells has not been reported.The phenomenon that tumor cells tend to rely on the glycolytic pathway to obtain energy even under aerobic conditions is called “Warburg effect” or aerobic glycolysis.It has been listed as one of the top ten features of tumors.Studies have shown that aerobic glycolysis is closely related to tumor proliferation and invasion.Combined with previous studies,we speculated that miR-3189-3p may affect the biological characteristics of tumor cells by affecting glucose metabolism.Taken together,this study investigated the effect of miR-3189-3p on the biological function of GC cells MGC803;and further studied whether miR-3189-3p participates in the effects of S100A4 on the biological characteristics of GC cells.The target genes of miR-3189-3p were predicted by bioinformatic software and validated related experiment.We further investigated whether miR-3189-3p influences the biological characteristics of GC cell MGC803 by its target gene CFL2.We also evaluated the prognostic value of CFL2.Finally,we explored the role of miR-3189-3p and target gene CFL2 in the glycolysis of GC cells.It is hoped that this study will be helpful to understand the effects of miR-3189-3p on the biological function of S100A4,and the underlying molecular mechanism,and provide new evidence and clues for the study of diagnosis and the treatment of GC by targeting S100A4.Materials and methods: 1.Materials: human GC cell line MGC803,human embryo kidney cell line HEK293 T.2.Methods: cell culture,transient gene transfection method,Real-time quantitative PCR assay,CCK-8 assay,Transwell assay,Scratch assay,sphere-forming assay,soft agar colony-forming assay,Western blotting detection,dual luciferase reporter assay,Lactate level test,Lactate dehydrogenase activity test,survival analysis.Results: 1.The results of Real-time quantitative PCR showed that at 48 hours after transfection of MGC803 cells with S100A4-siRNA,the expression of GDF15 mRNA,pri-miR-3189 and miR-3189-3p were significantly down-regulated compared with NC-siRNA group.2.The results of CCK8 assay showed that miR-3189-3p inhibitor effectively promote the proliferation of MGC803 cells;whereas,overexpression of miR-3189-3p by the transfection of miR-3189-3p mimics effectively inhibit the proliferation of MGC803 cells.3.The results of transwell and scratch experiments showed that overexpression of miR-3189-3p can effectively inhibit the migration of MGC803 cells.4.The results of sphere-forming and soft agar colony-forming assay showed that overexpression of miR-3189-3p can effectively inhibit the CSC-like properties of MGC803 cells.5.The overexpression of miR-3189-3p significantly enhanced the inhibitory effect of S100A4-siRNA on the proliferation,migration and CSC-like properties of MGC803 cells.6.Bioinformatic software predicts that there is a miR-3189-3p binding site in the 3'-UTR region of CFL2.7.The results of dual luciferase reporter assay showed that overexpression of miR-3189-3p significantly suppressed the luciferase activity of the wild-type CFL2 3'-UTR.8.Real-time quantitative PCR and Western blotting results showed that the overexpression of miR-3189-3p reduced the expression of CFL2 mRNA and protein compared with the control group.9.Real-time quantitative PCR and Western blotting results showed that the expression of CFL2 mRNA and protein were significantly down-regulated in MGC803 cells transfected with CFL2-siRNA compared with the control group,indicating that CFL2-siRNA could effectively inhibit the expression of CFL2.10.The silencing of CFL2 can effectively inhibit the proliferation and migration of MGC803 cells.11.The transfection of CFL2 expression vectors can effectively reverse the effect of miR-3189-3p on the proliferation and migration of MGC803 cells.12.Kaplan-Meier plotter predicts that high expression of CFL2 is closely related to poor prognosis of the patients with gastric cancer.13.The overexpression of miR-3189-3p in MGC803 cells reduces the level of lactate,inhibit the activity of lactate dehydrogenase.14.Real-time quantitative PCR results showed that at 72 hours after transfection of miR-3189-3p mimics,the expression of some glycolysis-related genes changed significantly compared with control group.15.The overexpression of CFL2 in MGC803 cells increased the level of lactate and promote the activity of lactate dehydrogenase.16.The results of real-time quantitative PCR showed that the expression of glycolysis-related genes didn't show significant change at 48 hours after transfection of CFL2 expression vector.Conclusions: 1.S100A4 gene silencing reduces the expression of miR-3189-3p in MGC803 cells.2.MiR-3189-3p inhibits the proliferation,migration and CSC-like properties of MGC803 cells.3.MiR-3189-3p enhances the inhibitory effect of S100A4-siRNA on the proliferation,migration and stem cell-like properties of MGC803 cells.4.CFL2 is a direct target gene of miR-3189-3p,mediating the effect of miR-3189-3p on proliferation and migration properties of GC cells.5.MiR-3189-3p inhibits glycolysis,while CFL2 promotes glycolysis of MGC803 cells.