Effect Of MicroRNA-27a-3p On The Proliferation And Invasion Of Gastric Cancer Cells And Its Mechanism

BackgroundMicroRNA is one kind of endogenous non-coding short-stranded RNA widely found in eukaryotic cells.miRNA is about 22-25 nt long,and regulates gene expression by targeting and binding target gene mRNA at the post-transcriptional level,and participates in the occurrence and progression of tumors.The mining and analysis of the database showed that the expression of miR-27a-3p was abnormally increased in gastric cancer cells.SIKl is a serine/threonine protein kinase,which belongs to the AMP-activated protein kinase family,and has a tumor suppressor effect in gastric cancer.As a member of the F-box family,FBXW7 mediates the ubiquitination modification and degradation of oncoproteins and is an important tumor suppressor protein.The bioinformatics website predicts that SIK1 and FBXW7 are the potential downstream target genes of miR-27a-3p.This project studies the effects of miR-27a-3p on gastric cancer cells,and explores whether miR-27a-3p exerts corresponding cytological effects through targeted regulation of SIK1 and FBXW7.MethodsFirst,using R language analyzes data of gastric adenocarcinoma from the TCGA database to find out the differences in the expression levels of miR-27a-3p between gastric cancer tissues and paired adjacent cancers;lipofectamineTM2000 was used to transfect miR-27a-3p mimics and miR-27a-3p NC into AGS cells,qPCR was used to detect the expression of miR-27a-3p in the experimental group and the control group.The CCK8 assay was performed to detect cell proliferation viability,the cell clone formation assay was used to detect cell cloning ability,and the Transwell assay was used to detect the ability of cell invasion.Searching on TargetScan website to predict the downstream target of miR-27a-3p in gastric cancer cells,and select SIK1 as the first downstream target gene for verification.Western blot assay was performed to detect the expression of SIK1 in the two groups of cells.FBXW7 was selected as second candidate gene,and dual luciferase reporter gene analysis was performed to verify the targeting relationship between miR-27a-3p and FBXW7.qPCR and Western bolt methods were separately used to detect FBXW7 mRNA and protein expression levels in cells.Data of Gastric adenocarcinoma was downloaded from TCGA database.Subsequently,using R language to analyze the difference of FBXW7 mRNA expression level between normal tissues and gastric cancer,and analyze the relationship between FBXW7 mRNA expression level and the 5-year survival rate.The SPSS 19.0 was used for data analysis,and differences between groups were compared by t-test.Results1.In the TCGA database,the relative expression of miR-27a-3p increased significantly in gastric cancer tissues compared with matched neighboring normal tissues.2.After transfection with miR-27a-3p,the expression of miR-27a-3p in the experimental group was significantly higher than that in the control group.CCK8 experiment,plate cloning experiment and Transwell invasion experiment showed that overexpression of miR-27a-3p can significantly enhance the cell proliferation activity,cloning ability and invasion ability of AGS gastric cancer.3.The bioinformatics website predicts that the SIK1 gene may be the target gene of miR-27a-3p.Overexpression of miR-27a-3p in AGS gastric cancer cells cannot down-regulate SIK1 protein expression.4.The FBXW7 gene was predicted and screened out as a candidate target gene of miR-27a-3p on the bioinformatics network.Overexpression of miR-27a-3p can inhibit the activity of wild-type FBXW7 3’UTR dual luciferase.After designing and changing its binding site,the inhibitory effect disappears.The mRNA and protein expression of FBXW7 in AGS gastric cancer cells overexpressing miR-27a-3p was significantly decreased.5.TCGA database analysis shows that the expression of FBXW7 mRNA in gastric cancer tissues is significantly lower than that in normal tissues,and the 5-year survival rate of the low expression group is lower.ConclusionsThe expression of miR-27a-3p is up-regulated in gastric cancer tissues,and the overexpression of miR-27a-3p in AGS gastric cancer cells can promote cell proliferation and invasion.In AGS gastric cancer cells,miR-27a-3p cannot target down-regulation of SIK1 protein expression.miR-27a-3p may enhance the proliferation and invasion ability of AGS gastric cancer cells by targeting down-regulation of the expression of tumor suppressor gene FBXW7.