The Effect And Mechanism Of LncRNA KCNQ1OT1/miR-4458/P53 Axis On Autophagy And Apoptosis Of Human Bronchial Epithelial Cells

Background:Chronic obstructive pulmonary disease(COPD),a common preventable and treatable disease,is characterized by persistent respiratory symptoms and incomplete reversible restricted airflow.COPD is currently the third leading cause of death in the world,with about 3.2 million patients dying of COPD each year.One of the main reasons for the high mortality of COPD is its high prevalence.The latest epidemiological survey shows that the prevalence of COPD in adults over 40 years old in China is 13.6%.At present,the main standard for diagnosis of COPD is pulmonary function examination.The main therapeutic drugs are bronchodilators and inhaled corticosteroids(ICSs),but the early diagnostic rate and treatment effect are unsatisfactory.Therefore,it is importance to explore the pathogenesis of COPD and further develop more effective diagnostic markers and therapeutic targets.Autophagy is an evolutionary conserved lysosomal degradation pathway that degrades damaged organelles and proteins.Autophagy is usually referred to as microautophagy,which is characterized by the formation of autophagosomes.In recent years,numerous studies have shown that autophagy plays an important role in the pathogenesis of COPD,but it is not clear whether autophagy is beneficial or harmful to COPD patients.The effect of smoking on autophagy of human bronchial epithelial cells(HBECs)is also still unclear.Some studies have shown that smoking can induce autophagy,and other studies shown that smoking can lead to defective autophagy.It is well known to all that smoking is one of the main pathogenic factors of COPD,so it is very important to study the effect of smoking on autophagy.Non-coding RNAs(ncRNAs)are a class of RNAs that do not encode functional proteins,among which microRNAs(miRNAs)and long non-coding RNAs(lncRNAs)have been widely studied.MiRNAs inhibit mRNA translation or promote mRNA degradation by binding the three prime untranslated regions(3’ UTR)of mRNA.LncRNAs act as miRNAs sponges and regulate gene expression by competitively binding miRNAs.Smoking can change the expression of miRNAs and lncRNAs,and miRNAs and lncRNAs play different roles by regulating autophagy.Studies have pointed out that ncRNAs may be a potential diagnostic markers and therapeutic targets of COPD.MiR-4458 can inhibit the proliferation and migration of tumor cells,and is a type of tumor suppressor.LncRNA KCNQ10T1 is highly expressed in ischemic stroke,myocardial infarction and a variety of pulmonary inflammatory diseases,and participates in the regulation of autophagy and apoptosis.However,the role of miR-4458 and KCNQ1OT1 in COPD is still unclear,so this study aims to explore the role and mechanism of miR-4458 and lncRNA KCNQ10T1 in COPD.Part Ⅰ MiR-4458 is predicted to be involved in the pathogenesis of COPD by sequencing and bioinformaticsObjective:Prediction of miR-4458 as autophagy-related miRNA and the mechanism of miR-4458 in COPD.Methods:1.Autophagy was induced by lipopolysaccharide(LPS)in HBECs,and miRNAs were obtained by transcriptome sequencing of cells in control group and LPS-stimulated group.2.R language was used for differential analysis of miRNAs obtained by sequencing,Targetscan,miRDB,and microT-CDS databases were used to predict the target genes of differential miRNAs.Target genes were intersected with autophagy-related genes(ARGs)from HADb database.The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment analyses of the intersecting genes were performed to determine whether the differential miRNAs were autophagy-related miRNAs.3.The genes expression levels were verified in lung tissue dataset GSE100281.GO and KEGG functional analyses and Protein-protein interaction(PPI)analysis of differentially expressed genes were also performed to predict whether differential expressed miRNAs were involved in the pathogenesis of COPD.Results:1.LPS could increase the autophagy level of HBECs,and 3754 miRNAs were obtained by transcriptomic sequencing.2.A total of 3621 target genes of 21 differential expressed miRNAs were predicted using three databases.55 target genes were obtained by intersecting with ARGs.Functional enrichment analysis showed that 55 target genes were mainly involved in autophagy,and 21 miRNAs were predicted to be autophagy-related miRNAs.3.20 differentially expressed target genes were identified by GSE100281.Functional enrichment analysis showed that 20 differentially expressed target genes were involved in autophagy,and four genes were identified as hub genes by PPI analysis.P53 is involved in regulating both the PI3K/AKT signaling pathway and apoptosis,miR-4458 may be involved in the pathogenesis of COPD through targeting P53 to regulate autophagy and apoptosis.Conclusions:MiR-4458 is an autophagy-related miRNA,and miR-4458 may play a key role in the pathogenesis of COPD by targeting P53.Part Ⅱ MiR-4458 alleviates CSE-induced autophagy and apoptosis of human bronchial epithelial cells by targeting P53Objective:To elucidate the expression level and mechanism of miR-4458 in COPD.Methods:1.The levels of miR-4458 in cigarette smoke extract(CSE)-exposed HBECs and peripheral blood mononuclear cells(PBMCs)of COPD patients were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR).2.HBECs were transfected with negative control(NC-mimics,NC-inhibitor),miR-4458 mimics,and miR-4458 inhibitors,respectively.The protein levels of autophagy and apoptosis markers were detected by western blotting(WB),cell apoptosis rate was detected by flow cytometry,CCK-8 and EdU proliferation assays were used to detect cell proliferation.3.P53 mRNA expression levels in CSE-exposed HBECs and PBMCs of COPD patients were detected by RT-qPCR.After transfection with NC-mimics,miR-4458 mimics,NC-inhibitor and miR-4458 inhibitor,respectively,the expression levels of P53 in CSE-exposed HBECs were detected by WB,and then the targeting relationship between miR-4458 and P53 was determined by luciferase reporter assay.4.HBECs were transfected with NC-mimics,miR-4458 mimics,NC-inhibitor and miR-4458 inhibitor,respectively,the protein levels in AKT/mTOR pathway were detected by WB.5.HBECs were transfected with NC-mimics or miR-4458 mimics and empty plasmid or P53 over-expression plasmid,respectively.The expression levels of autophagy and apoptosis markers,P53,and AKT/mTOR pathway protein were detected by WB,cell apoptosis rate was detected by flow cytometry,and cell proliferation was detected by CCK-8 and EdU proliferation assays.Results:1.Compared with the control group,miR-4458 was down-regulated in CSE-exposed HBECs and PBMCs of COPD patients.2.Over-expression of miR-4458 attenuated autophagy and apoptosis in CSE-exposed HBECs and enhanced cell proliferation,while knockdown of miR-4458 led to the opposite results.3.P53 was increased in CSE-exposed HBECs and PBMCs of COPD patients,the expression of P53 was decreased after over-expression of miR-4458,and increased after knockdown of miR-4458.Luciferase reporter assay showed that P53 was a direct target gene of miR-4458.4.The expression of p-AKT and p-mTOR were increased after over-expression of miR-4458,and the opposite results were obtained after knockdown of miR-4458.5.Over-expression of miR-4458 partially reversed the enhanced autophagy and apoptosis,decreased cell proliferation and inhibition of AKT/mTOR pathway induced by over-expression of P53 in CSE-exposed HBECs.Conclusions:MiR-4458 is down-regulated in CSE-exposed HBECs and PBMCs of COPD patients.MiR-4458 alleviates CSE-induced autophagy and apoptosis through the P53/AKT/mTOR pathway,it may be a promising COPD treatment target.Part Ⅲ LncRNA KCNQ1OT1 enhances CSE-induced autophagy and apoptosis of human bronchial epithelial cells by competitive binding of miR-4458Objective:To elucidate the expression level and mechanism of lncRNA KCNQIOT1 in COPD.Methods:1.LncBase and ENCORI databases were used to predict lncRNAs targeting miR-4458,and KCNQ1OT1 with the highest score was selected to further verify.2.The expression of KCNQ1OT1 in HBECs was knocked down by small interfering RNA(siRNA),and the knockdown efficiency was verified by RT-qPCR.WB was used to detect the expression levels of autophagy and apoptosis markers,flow cytometry was used to detect cell apoptosis rate,CCK-8 and EdU proliferation assays were employed to detect cell proliferation.3.After knockdown of KCNQ1OT1,the mRNA expression levels of miR-4458 in HBECs were detected by RT-qPCR,and the expression level of P53 protein was detected by WB.Then luciferase reporter assay was used to determine whether there is a direct targeted relationship between KCNQ10T1 and miR-4458.4.HBECs were transfected with NC-inhibitor,miR-4458 inhibitor,and miR-4458 inhibitor with si-KCNQ1OT1,respectively,autophagy and apoptosis markers expression levels were detected by WB,cell apoptosis rate was detected by flow cytometry,CCK-8 and EdU proliferation assays were used to detect cell proliferation.Results:1.23 target lncRNAs were obtained by intersecting predicted results of LncBase and ENCORI databases,the prediction score of KCNQ1OT1 was the highest.Compared with the control group,the KCNQ10T1 expression was up-regulated in CSE-treated HBECs and PBMCs of COPD patients.2.After KCNQ10T1 was knocked down,autophagy and apoptosis of HBECs were reduced,and cell proliferation was enhanced.3.Knockdown of KCNQ10T1 upregulated miR-4458 expression and downregulated P53 expression.Luciferase reporter assay showed a direct targeted relationship between KCNQ1OT1 and miR-4458.4.Knockdown of miR-4458 could partially reverse the decreased autophagy and apoptosis,enhanced cell proliferation and down-regulated of P53 expression caused by KCNQ1OT1 knockdown.Conclusion:LncRNA KCNQ1OT1 is highly expressed in CSE-exposed HBECs and PBMCs of COPD patients.KCNQ1OT1 promotes autophagy and apoptosis and inhibits cell proliferation by targeting miR-4458 in CSE-exposed HBECs.KCNQ1OT1 may be a potential therapeutic target for COPD.