Construction And Validation Of Long Non-coding RNA Mediated CeRNA Regulatory Network In T2 Asthma

BackgroundBronchial asthma is a chronic airway inflammatory disease mediated by a variety of inflammatory cells and pro-inflammatory factors,characterized by reversible airflow restriction,airway hyper-responsiveness and airway remodeling.In recent years,the incidence of asthma increases year by year,according to statistical models predict that the world’s asthma patients will reach 400 million people by 2025,which undoubtedly poses a serious threat to personal health,and increased the medical burden of each country.Asthma can be divided into T2 type asthma and non-T2 type asthma,among which T2 type asthma occupies a large proportion of asthma patients,and has a great effect on conventional therapy,such as inhaled corticosteroids,bronchodilators,etc..However,the current diagnostic criteria for T2 type asthma have not yet been unified and the treatment effects of patients with T2 asthma are divergent.Therefore,it is particularly significant to study the pathogenesis of T2 asthma and find more accurate biomarkers and personalized treatment.Long noncoding RNAs(lncRNAs)are RNA molecules with a length of over 200 bases without protein-coding potential.They were originally considered "transcriptional noise"since not involved in protein-coding.With the development of human genome sequencing,numerous transcriptome genes,especially lncRNAs,were reported to contain miRNA binding sites,thus leading to the proposal of the competing endogenous RNA(ceRNA)hypothesis.Non-coding RNAs(ncRNAs)(e.g.,lncRNAs,pseudogenes and circular RNAs)could serve as a sponge for miRNAs via miRNA response elements to have an indirect effect on the expressions of protein-coding mRNAs.According to the ceRNA hypothesis,the functions of many characteristically unknown lncRNAs are elucidated.However,the position and mechanism of the lncRNA-meditated ceRNA network involved in T2 asthma remains unclear.Objective1.To explore the differential expression of lncRNA,miRNA and mRNA in T2 asthma,search for biomarkers with diagnostic significance,and construct the ceRNA network relationship between the above-mentioned differential genes.2.To verify the diagnostic biomarkers and ceRNA network deduced from the bioinformatic analysis experimentally.MethodsMultiple sets of T2 asthma airway biopsy transcription profiles were collected,which involved long non-coding RNA(lncRNA),mRNA,and microRNA(miRNA).DIANAlncBase,targetscan,mirwalk,and miRDB databases were employed to predict interactions between lncRNAs,miRNAs and target mRNAs.To identify mRNAs correlated with T2 asthma,differential expression and network analyses were conducted through weighted gene coexpression network analysis(WGCNA).Subsequently,the expressions of potential biomarkers were examined through qRT-PCR analysis in the T2 asthma core interacting cellular factor(IL13/IL-33)induced experimental model.Lastly,the ceRNA network was confirmed by plasmid transfection and RNAi experiments in a 16HBE cell line.Results1.A total of 30 lncRNAs,22 miRNAs and 202 mRNAs were found to be significantly different in T2 asthma.A ceRNA network composed of 13 lncRNAs,12 miRNAs and 8 mRNAs was constructed by using the intergene relationships and upstream and downstream regulatory relationships.PCAT19 had the highest AUC value in ROC analysis between T2 and non-T2 asthma.2.The results of RT-PCR showed that the downstream molecular expression of 16HBE cells transfected with representative molecules was consistent with the results of biogenic analysis.Conclusions1.PCAT19 can be used as a biomarker for the identification of T2 asthma.2.We constructed and verified the presence of lncRNA-mediated ceRNA networks in T2 asthma.