MiRNA-206 Functions As An Anti-oncogenic MiRNA In Esophageal Squamous Cell Carcinoma By Suppressing C-Met/PI3K/AKT/mTOR Pathway

BackgroudEsophageal cancer(esophageal carcinoma EC)is the sixth most common malignancy in the worldwide.And EC is characterized by its high mortality rate,with a less than 15%5-year survival rate.In recent years,its incidence increased year by year.In the EastAsia,especially in China,Esophageal squamous cell carcinoma(ESCC)is the predominant pathological type of esophageal cancer,about 90%of the diagnosed esophageal cancer patients.Although the improvement in the early diagnosis and treatment of the disease such as resection and chemotherapy and surgical technique,has been made,the prognosis of ESCC patients remains poor.Therefore,it is urgent to dissect tumorigenesis mechanism of ESSC and explore the feasible therapeutic targets of ESCC.Understanding the mechanism of the occurrence and development of esophageal squamous cell carcinoma at the molecular level and exploring the diagnosis and effective treatment targets for early diagnosis of esophageal cancer and treatment have profound significance.MicroRNAs(miRNAs)are a class of 19-22-nucleotide non-coding RNAs that affect multiple pathways by binding to specific sites at 3’-UTR of target mRNAs.MiRNAs play an important role in regulating cellular differentiation,proliferation,aggressiveness,angiogenesis and apoptosis.Abnormity of miRNAs are closely related to the occurrence of a variety of tumors.More and more studies have found and confirmed that miRNAs is closely related to the occurrence and development of various human diseases,especially malignant tumors.Many kinds of miRNAs are involved in the occurrence and development of esophageal cancer.Some of them are regarded as oncogenes,while others serve as tumor suppressor genes.It may be possible to diagnose and treat esophageal squamous cell carcinoma by early intervention of miRNA.In this study,we first analyzed the expression level of miRNAs in ESCC tissue by microarray technology.Among aberrantly expressed miRNAs,miR-206 has aroused our interest.MiR-206 functions as a tumor suppressor gene in many kinds of tumors,but it has not been reported in ESCC.Therefore,we select miR-206 for further study and determine the expression of miR-206 in ESCC tissues and cell lines.We analyzed the relationship between the abnormal expression of miR-206 and clinicopathologic features.The expression of miR-206 was up-regulated after miR-206 mimics transfection and observed the alteration of the proliferation and apoptosis of ESCC cells.Moreover,we search for the direct target genes of miR-206 through dual-luciferase reporter assays and measured the expressions of the related signal pathway regulated by miR-206.Our findings provide a new theoretical basis for the diagnosis and treatment of ESCC.Part 1 miR-206 expression is downregulated in ESCC tissues and cell lines,and correlated with clinicopathologic featuresObjectiveTo investigate miR-206 expression obtained from microarray in human ESCC tissue and cell lines,analyze the relationship between miR-206 abnormal expression and clinical features,such as differentiation,lymph node metastasis,and TNM stage.MethodsThe study included 52 patients who were first diagnosed with ESCC in the Department of Thoracic Surgery,Second Affiliated Hospital of Zhengzhou University from October 2010 to September 2016.ESCC tissues and adjacent normal tissues were collected from patients after surgical resection.Quantitative real time-PCR was performed to test miR-206 expression levels in 52 ESCC tissues and matched adj acent normal tissues.We also assessed miR-206 levels in four ESCC lines.Results1 Microarray assay reveals that 35 miRNAs were upregulated and 22 miRNAs were downregulated.Among the differentially expressed miRNAs,miR-206 was one of the most downregulated miRNAs.2.The level of miR-206 expression in ESCC tissues was significantly lower than that in normal tissues.In accordance with clinical data,the expression of miR-206 was obviously downregulated in four ESCC cell lines(Ec9706,Eca109,TE13 and KYSE410)as compared to Het-1A.3 Low expression of miR-206 was inversely associated with lymph node metastasis,TNM stage and.However,there were no significant correlations between miR-206 expression level and gender,age,tumor location,differentiation or distant metastasis in ESCC.Kaplan-Meier survival analysis revealed that patients with low miR-206 expression had shorter overall survival.Part 2 Overexpression of miR-206 inhibited cell proliferation and induced cell apoptosisObjectiveObserve the effects of miR-206 on the proliferation and apoptosis of the cell lines.MethodsTo investigate the potential functions of miR-206 in ESCC cells,Eca109 and KYSE410 cells,with lowest expression in four cell lines,were transfected with the miR-206 mimic or mimics-NC,then CCK-8 assay used to evaluate the effect of miR-206 on ESCC cells’ proliferation ability.Apoptosis assay to measure cell apoptosis.Results1 The expression level of miR-206 in Eca109 and KYSE410 cells was significantly increased after transfection.2 CCK-8 assay showed that the cell proliferation was significantly inhibited in miR-206-mimic-transfected Eca109 and KYSE410 cells compared with mimics-NC-transfected cells.3 Flow cytometer analysis showed that the cell apoptosis was markedly promoted in miR-206-mimic-transfected Eca109 and KYSE410 cells compared with mimics-NC-transfected cells.Part 3 The preliminary investigation of the mechanism of miR-206 in ESCCObjectiveIt is now clear that miRs execute their function by regulating the expression of target genes.By using bioinformatics software,we predicted that miR-206 has multiple potential target genes.Among them,c-Met(also known as MET)a receptor for hepatocyte growth factor(HGF),aroused our interest,which has been found to function as an oncogene.Thus we chose c-Met to conduct further research and to explore whether c-Met is the target gene of miR-206 to influence the biological behavior of ESCC cells.MethodDual luciferase experiment was performed to verify whether c-Met is a direct target gene of miR-206.We further verify whether the abnormal expression of c-Met was mediated the anti-tumor effects of miR-206 on ESCC cells.Western Blot was used to detect the effect of miR-206 on AKT/mTOR signal pathway.Result1 Luciferase reporter gene assay showed that overexpression of miR-206 dramatically repressed,while knockdown of miR-206 increased the relative luciferase activity of constructs containing the wild-type c-Met 3’UTR.However,the luciferase activity of the reporter containing the mutant binding site has no obvious change.2 We also found that overexpression of miR-206 significantly inhibited,whereas knockdown of miR-206 promoted the c-Met expression on protein level.3 Overexpression of c-Met partly reversed the inhibitory effects of miR-206 on the cell proliferation in Eca109 and KYSE410 cells.We also showed that overexpression of c-Met abrogated the promoting effects of miR-206 on cell apoptosis in Eca109 and KYSE410 cells4 Overexpression of miR-206 significantly inhibited the phosphorylation of Met,AKT and mTOR,indicating that miR-206 inhibited the activation of AKT/mTOR signaling pathway.