Bioinformatics Analysis Of Benign Airway Stenosis And Mechanism Of Action Of Circ-0130687/miR-139-5p/AKT3 Axis In Benign Airway Stenosis

Benign airway stenosis lesions are mainly located in the central airways such as the trachea,left and right main bronchi,and right middle bronchi,and central airway stenosis are caused by granulation tissue and scarring is the most common.Patients may have different respiratory manifestations,and in severe cases,respiratory failure may lead to death.Benign airway stenosis are caused by susceptibility genes,local mechanical compression,multiple infections of the trachea and lungs and other factors lead to the release of various cells or inflammatory response factors,fibroblasts are recruited to the wound site,some fibroblasts undergo fibrogenesis into myofibroblasts,myofibroblasts promote wound contraction,and eventually form scarring,the essence of benign airway stenosis is fibroproliferative diseases.Although there are many treatments for benign airway stenosis,benign airway stenosis has become a problem in the field of respiratory intervention due to its high restenosis rate.At the same time,the mechanism of benign airway stenosis is still being explored,so it is urgent to take effective preventive measures to inhibit its progression.Circular RNA(circ RNA)is a new type of non-coding RNA first discovered by humans in the 70 s.It has multiplexity,high stability,richness of expression and permeability.Circ RNA is the main regulator of gene expression and can regulate gene expression in a variety of ways,especially as a mi RNA sponge,which inhibits the expression,transcription and splicing of target genes by binding competitively to mi RNA.The role of circ RNA in cancer,cardiovascular disease,neurological disease,osteoarthritis and other diseases cannot be ignored.In addition,in recent years,more and more studies have found circ RNA to be involved in the process of pulmonary fibrosis.Unfortunately,the function of circ RNA in benign airway stenosis can not be specified.Therefore,in-depth study of circ RNA and its molecular mechanism of benign airway stenosis fibrosis can provide a new perspective for the diagnosis and treatment of benign airway stenosis,which is of great clinical significance.This study aims to explore the expression profile of circ RNA in peripheral venous blood of patients with benign airway stenosis,screen differentially expressed circ RNA,and further explore the main functions and possible molecular mechanisms of circ RNA in in vitro cell experiments.The specific experiments of this study include the following three parts:Part One Expression profiling of circular RNA in peripheral blood inpatients with benign airway stenosisObjective: The expression profile of circ RNA in peripheral blood of patients with benign airway stenosis was obtained by high-throughput sequencing technology,circ RNA with differential expression was screened,and circ RNA with benign airway stenosis was preliminarily determined to be significantly different from the normal control group.Methods: Patients with benign airway stenosis confirmed by transcervicothoracic CT and bronchoscopy were recruited into the lesion group(n=6),and those with no abnormalities in normal physical examination were recruited into the normal control group(n=5).The differentially expressed circ RNA and m RNA in whole blood of 6 patients with benign airway stenosis and 5 normal control groups were analyzed by high-throughput sequencing.The differentially expressed circ RNA and m RNA genes in the transcriptome of lesion samples and normal control samples were screened using DESeq2 pack,volcanoes were plotted using R ggplot2 to show the expression of differential genes,and R-language pheatmap was used to plot the expression calorimetry of differential genes.Results: Compared to the normal control group,583 differentially expressed circ RNAs were screened,of which 434 circ RNAs were upregulated and 149 circ RNAs were downregulated.At the same time,229 differentially expressed m RNAs were screened,of which 146 m RNAs were upregulated and83 m RNA were downregulated.Circ RNA is mostly derived from exons,and most circ RNAs have 2-6 exons.The distribution of the number of circ RNAs produced by genes shows that only one circ RNA is produced by most genes.Conclusions:1.Circ RNA is mostly derived from exons,and only one circ RNA is produced by most genes.2.This study revealed the differential expression of circ RNA in peripheral venous blood of patients with benign airway stenosis,and provided a rich circ RNA and m RNA database for the screening of further key genes and the construction of ce RNA networks.Part Two Construction Ce RNA network of benign airway stenosis andscreening of key genesObjective: Differentially expressed circ RNA and m RNA were used to construct ce RNA regulatory networks significantly associated with benign airway stenosis,and key genes were screened.Methods: The Rstudio cluster Profiler package was used to show GO and KEGG enrichment analysis of differentially expressed genes,and using the enrichplot package to plot bubble plots to visualize the enrichment results.Construct a protein interaction network for differentially expressed m RNA genes by the STRING website.The protein network diagram is then beautified with Cytoscape software.The key genes are obtained using the Cyto Hubba plug-in in Cytoscape software.Cytoscape’s plug-in Clue GO performed enrichment analysis of the key genes.The pearson correlation of key m RNAs was obtained by PPI network analysis with cric RNAs was used to build m RNA-cric RNA co-expression networks,and the results were optimized by Cytoscape software.Mi RNA-m RNA pairs were obtained by screening mi RNAs who were associated with m RNA genes through the starbase database.The two sets of relationships were paired to construct a circ RNAmi RNA-m RNA network.The results were optimized by Cytoscape software.Results: Enrichment analysis of differentially that expressed m RNA showed that it was mainly enriched in biological processes such as neutrophil degranulation,neutrophil activation involved in immune response,neutrophilmediated immunity,and neutrophil activation.PPI network analysis obtained160 protein interaction network relationships,and further screening obtained46 key genes.The TF-m RNA regulatory network was constructed by 46 key genes includes 274 nodes and 573 relationship pairs,and 251 potential therapeutics exist in the DGIDB database.Classical pathway analysis of 46 key genes showed that it was mainly involved in sepsis signaling pathway,cellular immune response and HIF-1 signaling pathway.At the same time,30 circ RNAs,36 m RNAs and 275 mi RNAs constitute a circ RNA-mi RNAm RNA network.Conclusions:1.In the study,229 differentially expressed m RNAs were found to be associated with oxygen transport,neutrophil activation,immune response and oxygen binding.2.The use of IPA analysis found that key genes were highly correlated with inflammatory activation and acute respiratory distress syndrome.3.Constructing circ RNA-mi RNA-m RNA network was used to lay a theoretical foundation for further in vitro cell experiments.Part Three Study on the mechanism of action of circ-0130687/mi R-139-5p/AKT3 axis in benign airway stenosisObjective: The specific mechanism of circ-0130687/mi R-139-5p/AKT3 axis in benign airway stenosis was further explored.Methods:Human airway fibroblasts were selected for cell experiments,cell induction technology was applied,airway fibroblasts were induced at different concentrations of TGF-β1 at different times,simulating the living environment of airway fibroblasts in patients with benign airway stenosis,and the expression of circ-0130687 in cells was verified by QPCR method,and cell proliferation after knocking down circ-0130687 was detected by cell counting test,cell scratch test and flow cytometry apoptosis method.For cell invasion migration ability and apoptosis rate,western blot was measured the expression level of AKT signaling pathway-related protein(AKT3)in each group of cells.The competitive relationship of ce RNA ternary pairs was validated by diluciferase reporter.Results: Circ-0130687 was expressed in both normal control and model groups,but significantly was increased in the model group.After knocking down circ-0130687,it inhibited the proliferation,migration and invasion ability of human airway fibroblasts and promoted apoptosis.Circ-0130687 downregulates mi R-139-5p activity,and mi R-139-5p interacts with AKT3 and downregulates AKT3 activity.Conclusions:1.In the benign airway stenosis model group,circ RNA and target protein expression were upregulated,while mi RNA was downregulated,circ RNA expression was positively correlated with target protein expression,and the target protein expression was negatively correlated with mi RNA expression.2.Circ RNA directly targets the regulation of mi RNA,circ RNA through mi RNA-139-5p/AKT3 axis is involved in the formation of benign airway stenosis.