MiR-127-5p Inhibits Glutamine Metabolism And Malignant Phenotype In Breast Cancer By Targeting MDH1

Breast cancer is the most common malignant tumor in gynecology in the world.The mortality and morbidity rate are the first among female malignant tumors,which have adverse effects on women’s health and quality of life.In recent years,although the diagnosis and treatment of breast cancer has made a big breakthrough,the five-year survival rate of breast cancer is still low,and the clinical treatment effect and prognosis of patients are poor.Therefore,elucidating the molecular mechanism of breast carcinogenesis and development is of great significance for the discovery of new breast cancer diagnostic markers and treatment stratigies.MicroRNAs(miRNAs)are small,non-coding RNAs of about 22-25 nucleotides in length that bind to the 3?untranslated regions(3?UTR)of the target gene mRNA.It negatively regulates the function of mRNA and thus participates in the regulation of various cellular biological functions.Recent studies have shown that miRNAs are involved in the regulation of the biological functions of various tumors including breast cancer.There is a large body of evidence that miR-127-5p is down-regulated in a variety of tumors and acts as a tumor suppressor,and studies have found that the miR-127-5p promoter is hypermethylated in breast cancer,indicating its tumor suppressor role in breast cancer,but its expression and regulation in breast cancer is still unclear.In this study,we first determined the down-regulation of miR-127-5p expression in breast cancer tissues and cells by real-time qPCR.Secondly,we found that overexpression of miR-127-5p by its mimics can notably suppress proliferation,invasion and enhance apoptosis of MDA-MB-231 cells,while down-regulation of miR-127-5p expression by its inhibitor can significantly promote the proliferation,invasion and suppress apoptosis of MCF-7 cells via cell proliferation,invasion and apoptosis assays.Furthermore,we predicted through multiple miRNA target gene prediction algorithms that MDH1 is a target gene of miR-127-5p,and further found that miR-127-5p can interact with the 3?UTR region of MDH1 by Dual-Luciferase Assay and down-regulate MDH1 protein expression by Western blot.These results indicate that MDH1 is a target gene regulated by miR-127-5p.MDH1(Malate dehydrogenase 1)is a key enzyme involved in the metabolism of glutamine,utilizing the NAD~+/NADH cofactor system to catalyze the reversible transformation of malic acid(Mal)and oxaloacetate(OAA),which plays an important role in the glutamine-dependent NADPH production process.Since the transformation between NADPH and NADP~+is accompanied by a transformation between glutathione oxidized(GSSG)and reduced(GSH)forms,MDH1 can affect the redox homeostasis of the cells.We overexpressed miR-127-5p in MDA-MB-231 cells and treated with miR-127-5p inhibitor in MCF-7 cells,and found miR-127-5p regulates glutamine metabolism and affects ROS levels in breast cancer cells by glutamine metabolism quantitative assay kit and ROS assay.In addition,we co-transfected miR-127-5p mimics and MDH1 plasmids in MDA-MB-231 cells and miR-127-5p inhibitor and siMDH1 in MCF-7 cells,and examined cell proliferation,invasion,and apoptosis,as well as glutamine metabolism and ROS levels.It was found that MDH1 is involved in the tumor suppressive effect of miR-127-5p on breast cancer cells.In a word,this study indicated that miR-127-5p functions as a tumor suppressor in breast cancer through targeting MDH1 and modulating the glutamine metabolism.Therefore,our results might provide a potential diagnostic marker and therapeutic target for breast cancer.