Construction Of A Prognosis-associated MicroRNA Risk Model For Lung Adenocarcinoma And Preliminary Study Of MiR-101-3p Tumor Suppressor Mechanism

Section1 Identification of a seven-microRNA signature for predicting survival in lung adenocarcinomaObjective:Lung cancer is the leading cause of cancer-related deaths worldwide.Although many achievements have been made regarding diagnosis and therapies in the past decades,the prognosis of lung cancer still remains poor.MicroRNAs(miRNAs)are small and short non-coding RNAs,which play an important role at the posttranslational level.Deregulated miRNAs were found to be involved in the initiation and progression of human cancers.Accumulating evidence indicated that miRNAs could be novel biomarkers in many aspects,including the prognostic biomarkers for cancers.The aim of this study was to explore cancer-specific miRNAs associated with the prognosis of lung adenocarcinoma(LUAD),which might lay the foundation of predicting survival and developing individualized treatment in LUAD.Methods:1.We downloaded the miRNA-Seq data and clinical information of 522 LUAD patients from the TCGA database,and identified the differentially expressed miRNAs between LUAD and adjacent normal tissues.2.We randomly assigned 522 LUAD patients into training set and testing set and developed a miRNA-signature for predicting patients' survival in the training set by LASSO&COX regression model.3.We further validated the miRNA-signature for predicting patients' survival in the testing and whole cohort,respectively.4.We identified the association between the miRNA-signature and other clinical factors by univariate and multivariate cox regression analyses.Results:1.72 differentially expressed miRNAs were identified between LUAD and adjacent normal tissues(fold change>2 and FDR<0.05),with 45 up-regulated miRNAs and 27 down-regulated miRNAs.2.7 survival-related miRNAs were identified in the training set and were developed as a prognostic model based on the expression of 7 miRNAs and their regression coefficient.Risk score=(-0.109xmiR-101-3p)+(-0.455×miR-148a-3p)+(0.146xmiR-192-5p)+(0.179xmiR-193b-3p)+(0.383xmiR-505-3p)+(0.212xmiR-584-5p)+(-0.06xmiR-99a-5p).3.The patients in the low-risk group had a longer overall survival than the high-risk group in the training set,testing set and the whole cohort,respectively(all P value<0.05).4.Risk score was considered as an independent factor by multivariate cox regression analysis in the training set,testing set and the whole cohort,respectively(training set:HR=1.97,P=0.02;testing set:HR=1.927,P=0.009;the whole cohort:HR=1.909,P=0.001).Conclusions:1.MiR-101-3p,miR-148a-3p,and miR-99a-5p were positively associated with the prognosis of LUAD patients;while miR-192-5p,miR-193b-3p,miR-505-3p,and miR-584-5p were negatively associated with the prognosis of LUAD patients.2.The patients could be divided into low-risk and high-risk groups according to the risk score model consisted of the 7-miRNA signature,which could be an independent prognostic factor.Section 2 MiR-101-3p acts as a tumor suppressor in lung adenocarcinoma by targeting TGFAObjective:Deregulated miRNAs play a vital role in human cancers,which may act as either oncogenes or tumor suppressor genes during the initiation and progression of tumors.Complex regulation networks comprised of miRNAs and target genes were involved in many biological processes.Accumulating evidence demonstrated that the expression level of miR-101-3p were deregulated in various cancers,including lung cancer.However,the function and potential mechanism of miR-101-3p remains to be further elucidated.Methods:1.We integrated the expression levels of miR-101-3p in LUAD and adjacent normal tissues by downloading the miRNA-Seq data from the TCGA and Gene Expression Omnibus(GEO)database.2.We detected the expression levels of miR-101-3p in BEAS-2B,A549,and H1299 cells by qPCR.3.To investigate the biological functions of miR-101-3p in LUAD,several assays(CCK-8,colon formation,wound healing assay,and transwell invation assay)were performed after transfection of miR-101-3p mimic or mimic-NC in A549 cells.4.The target genes of miR-101-3p were predicted by 4 miRNA target-predicted algorithms(TargetScan,miRanda,PITA,and RNA 22)and followed by gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis to find the optimal targeted gene.5.We further determined the mRNA and protein expression of TGFA gene by qPCR in A549 cells transfected with miR-101-3p mimic or mimic-NC.6.We identified the mRNA expression level of TGFA by TCGA database and analyzed the prognostic value of TGFA in LUAD patients by Kaplan-Meier plotter database.Results:1.Compared with normal lung tissues,the expression levels of miR-101-3p in LUAD tissues were all down-regulated among the TCGA and 3 GEO datasets(GSE74190,GSE51853,and GSE48414;all P value<0.001)2.Compared with BEAS-2B cells,the expression levels of miR-101-3p in A549 and H1299 cells were also down-regulated(P value<0.01).3.Cell proliferation,migration and invasion were significantly inhibited by overexpressed miR-101-3p(P value<0.05).4.TGFA was identified as the optimal targeted gene of miR-101-3p.The mRNA and protein expression levels of TGFA gene were significantly down-regulated by overexpressed miR-101-3p(P value<0.01).5.The mRNA expression level of TGFA was up-regulated in LUAD tissues,which was associated with poor prognosis(HR:1.44;P=0.0023).Conclusions:miR-101-3p inhibits lung adenocarcinoma cell proliferation,migration and invasion by suppressing TGFA gene expression.