MiRNA-32-5p Targets KLF2 To Promote Gastric Cancer By Activating PI3K/AKT Signaling Pathway

ObjectivesThe purpose of this study is to exploriedexploremiRNAs that can target KLF2 in gastric cancer cells,and to clarifiedclarify the molecular mechanism of KLF2 in regulating PTEN in gastric cancer.These studies provide theoretical and experimental basis for exploring gastric cancer biomarkers and targeted drug development.Methods(1)Construction of KLF2 overexpression and knockout gastric cancer cell line;(2)The bioinformatics software Targetscan was used to predict the miRNAs that can be combined with KLF2.After screening,the Dual-Luciferase Reporter Assay System was used to verify the regulation of miRNA on KLF2.MTS,Transwell and clone formation experiments were used to further verify the regulation of miRNAs on KLF2 at the cellular level,and observe the changes between proliferation and migration of gastric cancer cells;(3)Targetscan software was used to predict the binding site of KLF2 on the PTEN promoter,and then the Dual-Luciferase Reporter Assay System and Western blot were used to verify the regulation of KLF2 on PTEN;(4)The expression of PTEN and PI3K/AKT pathway related proteins was detected by Western blot after co-transfection of miRNA32-5p and shKLF2 in gastric cancer cells.Results(1)We successfully established stable KLF2 overexpression and knockout gastric cancer cells.In vitro experiments indicated thatknocking out KLF2 significantly promoted the proliferation and migration of MKN45 cells,while the results were reversed in MGC803 cells with KLF2 overexpression.(2)miRNA-32-5p was predicted and screened by bioinformatics software Targetscan as the research object.TCGA database analysis displayed thatthe expression of miRNA-32-5p was significantly increased in gastric cancer compared with normal tissues.Subsequently,the analysis of the luciferase reporter gene system revealed that the fluorescence activity of wild-type transfected miRNA-32-5p mimics was significantly reduced compared with the mutant group.Besides that,the results of Western blot demonstrated that miRNA-32-5p can inhibit expression of KLF2.In addition,the results of cell function experiments in vitro indicated that miRNA-32-5p and KLF2 gene knockout have a synergistic effect on promoting the proliferation and migration of gastric cancer cells.(3)Three binding sites of KLF2 in PTEN promoter were predicted by software Targetscan,and PTEN wild-type and mutant plasmids were constructed and transfected into MKN45 cells for fluorescence detection.The results displayed that compared with control group and mutant group,the fluorescence activity of wild-type cells was significantly lower Western blot results showed that the expression of PTEN was downregulated after KLF2 knockout and transfection of miRNA-32-5p mimics,while phosphorylation levels of AKT,mTOR and p70S6K were increased in gastric cancer cells.ConclusionIn summary,our experiments confirm that KLF2 directly binds to the PTEN promoter,and affects its expression at the transcription level,and proposed the binding site sequence of KLF2 acting on PTEN promoter for the first time.In addition,the selected miRNA-32-5p was proven to be involved in the development of gastric cancer by targeting KLF2 and PTEN,which enriches the theoretical basis of the molecular mechanism of gastric cancer occurrence and metastasis,and the basic work is of great significance for the clinical diagnosis and treatment of gastric cancer.Figure 15 table 10 reference 66...