The Study Of The Function And Molecular Mechanism Of MiR-585-5p On The Malignant Biological Behavior Of Gastric Cancer

Objectives Gastric cancer(GC)is the digestive tract tumor with high malignant degree,morbidity and mortality.The occurrence of gastric cancer is influenced by multiple factors and regulation.Micro RNA(miRNA)almost all participate in the occurrence and development of gastric cancer,which includes cells proliferation,apoptosis,epithelial mesenchymal transformation,vascularization,invasion and metastasis,and so on.The current report indicates that miR-585-5p has different abnormal expression in lung cancer,oral cancer and cardiovascular disease.Meanwhile,miR-585-5p with miR-218 plays a tumor suppressor role in oral cancer.However,the role of miR-585-5p in gastric cancer has been no relevant research reports.The present study aims to determine the effect of miR-585-5p in gastric cancer and analysis of miR-585-5p in the role of gastric cancer patients with malignant phenotypes and clinical prognosis.And further explore the development and progression of miR-585-5p mechanism,which will provide novel targets for the treatment of patients with gastric cancer.Methods1.The differential expression of miR-585-5p in normal gastric epithelial cell lines and gastric cancer cell lines was determined by Real-time PCR.In gastric cancer tissue arrays,the expression of miR-585-5p was detected through In situ hybridization(ISH),and its relationship between the prognosis of gastric cancer patients was studied.2.Mi R-585-5p mimics,inhibitors,overexpression and sh RNA lentiviral vectors were constructed.Through transient transfection and stable infection,miR-585-5p gainand loss-of-function cells were established.And roles of miR-585-5p in gastric cancer cells proliferation and metastasis were studied by MTT assay,Transwell migration assay and nude mice tail vein metastatic assay.3.Mi R-585-5p differential expression candidate target genes were selected by bioinformatics and Real-time PCR;The interaction relationship between miR-585-5p and candidate target genes was verified through double luciferase reporter gene assay;After up-or down-regulation of miR-585-5p,Western blot and q RT-PCR were applied to detect the expression changes of target genes;Through the function experiment and rescue assay,the target genes function and the influence of miR-585-5p in gastric by the target genes were verified.Results1.Mi R-585-5p expression was significantly reduced in m RNA level of gastric cancer cells.In tissue arrays,miR-585-5p expression was distinctly down-regulation compared with homologous normal tissues with statistical significance.Furthermore,Kaplan-Meier analysis revealed that patients with miR-585-5p down-regulation may have shorter survival time than those relative miR-585-5p up-regulation.2.MTT assays,Transwell migration assays and nude mouse transfer experiment indicated that miR-585-5p could suppress GC metastasis with no influence on the proliferation.3.MITF was selected as a candidate target gene for significantly differentially expressed in the help of bioinformatics software and q RT-PCR.Mi R-585-5p inhibited MITF expression by a directly targeting region with the analysis of luciferase reporter gene.Function experiment and rescue assay showed that MITF could promote the gastric cancer cell migration and MITF expression levels were visibly negative correlation between miR-585-5p by using q RT-PCR and Western blotting analysis.The MITF mutant expression construct without 3'-UTR of miR-585-5p could reverse miR-585-5p function of increasing migration.Immunofluorescence analysis discovered MITF mainly located in the cytoplasm.According to Immunohistochemistry,kaplan-Meier survival test showed that MITF expression was negatively associated with patients' survival.Conclusions In gastric cancer cells and tissues,we discovered the expression of miR-585-5p.Mi R-585-5p could suppress GC metastasis and had no relationship with proliferation. Patients with miR-585-5p down-regulation had shorter survival time than those relative miR-585-5p up-regulation.The study illuminated the tumour-suppressive function of miR-585-5p by directly targeting MITF.The patients with high expression of MITF had shorter survival time than those relative MITF low expression.