Effect And Mechanism Of MiR-146a On Malignant Biological Behaviors Of Lung Adenocarcinoma Cell Line

Background and ObjectiveAt present,the incidence and mortality of lung cancer remain high,accounting for the first place in global malignant tumors.Non-small cell lung cancer(NSCLC)accounts for about 80% of the total number of lung cancers.Lung adenocarcinoma is the most important and most common sub-type.Because lung cancer tends to be insidious and lacks specific symptoms,about 70% to 80% of patients in China are in advanced stage at the time of diagnosis,and it is difficult to perform radical surgery.Medical treatments with radiotherapy and targeted therapy are the main treatment.Because of its resistance,the efficacy is still unsatisfactory,and the prognosis is generally poor.Therefore,it is great significance to study the molecular biological mechanism of lung cancer development and explore new targets in the diagnosis and treatment of lung cancer.MicroRNAs(miRNAs)are a class of conserved endogenous non-coding RNAs that are about 22 nucleotides and are involved in many pathophysiological processes in the body.Studies have shown that there are many miRNA gene mutations in the development of lung cancer,and the expression of miRNAs were found in lung cancer tissues and adjacent tissues.In addition,some miRNAs play a special role in the development of lung cancer.MicroRNA-146a(miR-146a)is one of the most widely studied miRNAs.The role of miR-146 a in cancer,breast cancer,liver cancer and other tumors is relatively clear The role and mechanism of lung cancer remains to be studied.In our study,human lung adenocarcinoma cell lines were used as the research object to detect the expression of miR-146 a.The effects of miR-146 a on the malignant biological behavior of human lung adenocarcinoma cells were also investigated and explored.In addition,the target genes of miR-146 a were explored and mechanisms were studied to provide new potential targets for the diagnosis and treatment of lung adenocarcinoma.Methods:1.The expression of miR-146 a was detected by real-time fluorescence quantitative PCR(RT-qPCR)in three human lung adenocarcinoma(A549,H1299,PC-9)and human normal lung epithelial cells(BEAS-2B).2.Select appropriate human lung adenocarcinoma cell line as the research object;transfect miR-146 a mimics or inhibitor as experimental group,transfect miR-146 a mimics or inhibitor nonsense sequence as control group,the mock group only added the transfection reagen of lip2000.After 6 hours of transfection,the transfection efficiency was observed under fluorescence microscope.The expression multiple of miR-146 a was detected by RT-qPCR assay after 48 hours transfection.3.The effects of miR-146 a on the malignant biological behaviors such as proliferation,apoptosis,invasion and migration of human lung adenocarcinoma cells were detected by CCK8 assay,flow apoptosis assay,Transwell invasion assay and scratch assay.4.Using bioinformatics software(Targetscan and miRDB)to predict miR-146 a target genes,select suitable target genes by searching a large number of literatures,and dual-luciferase reporter gene was used to verify whether there was a targeted regulatory relationship between miR-146 a and target gene.5.RT-qPCR and Western blotting(WB)experiments were performed to verify the regulation of miR-146 a on target genes at messenger RNA(mRNA)and protein levels.Results:1.Compared with BEAS-2B cell line,miR-146 a was down-regulated in three human lung adenocarcinoma cell lines(A549,H1299 and PC-9).2.A549 cells were selected as experimental subjects,and transfected with miR-146 a mimics for 6 hours,the fluorescence transfection was observed and the approximate transfection efficiency was over 90%.After 48 hours,the over-expression of miR-146 a was detected by RT-qPCR to 10000 times.3.CCK8 assay,flow apoptosis assay,Transwell invasion assay and scratch assay showed that over-expression of miR-146 a could inhibit the proliferation of A549 cells,and promote the apoptosis of A549 cells,inhibit the invasion and migration of A549 cells.4.Through Targetscan and miRDB online predictive analysis,miR-146 a binds to 3'-UTR-related sequences of Interleukin Receptor-Associated Kinase 1(IRAK1)and TNF receptor associated factor 6(TRAF6).The results of dual luciferase reporter genes showed that the ratio of cell luciferase activity in the wild-type reporter gene vector(IRAK1-WT,TRAF6-WT)and mir-146 a mimics group was significantly reduced,while the ratio of cell luciferase activity in the mutated reporter gene vector(IRAK1-WT,TRAF6-WT)and miR-146 a mimics(or miR-146 a nc)group was not significantly difference.5.The results of RT-qPCR and WB experiments showed that over-expression of miR-146 reduced the expression of target genes(IRAK1 and TRAF6)in mRNA and protein levels in A549 cells.Conclusion:MiR-146 a is significantly reduced in human lung adenocarcinoma cells,which should play the role of tumor suppressor gene.MiR-146 a significantly inhibits the proliferation,growth,invasion and migration of A549 cells and promotes apoptosis,which may be achieved by inhibiting the expression of IRAK1 and TRAF6,which is expected to provide an effective new target for clinical molecular therapy of lung cancer.