Differential Expression Of CircRNAs In Idiopathic Pulmonary Fibrosis And Related Bioinformatics Analysis

Objective:Idiopathic pulmonary fibrosis(IPF)is a chronic,fatal interstitial lung disease with complex pathogenesis and poor prognosis,and currently there is no effective treatment.CircRNAs,as potential therapeutic targets and diagnostic biomarkers for some diseases,have become a hot spot in the field of RNA research in recent years.However,the mechanism of circRNA in pulmonary fibrosis remains unclear.In this study,the differential expression of circRNA in patients with idiopathic pulmonary fibrosis was investigated,and circRNA with obvious differential expression was screened for verification,providing a theoretical basis for the study of circRNA in pulmonary fibrosis disease.Methods:1.Lung tissues and normal lung specimens from 3 patients with idiopathic pulmonary fibrosis were collected,and the differentially expressed circRNA in the lung tissues of patients with IPF and normal lung tissues was detected by CIRI2 software.Hsa_circ₀001092,hsa_circ₀006354,hsa_circ₀026782,hsa_circ₀005328,hsa_circ₀000471,and hsa_circ₀002490 were selected as circRNA with significant difference expression.2.The experiment was divided into two groups: the experimental group(TGF-?1)and the control group(control).The experimental group was given the medium adding 5ng/ml TGF-?1,and the control group was given the medium adding the same volume for 72 h.The expression of A-SMA protein was detected by Western Blot.The expression level of the selected 6 genes in TGF-?1-induced lung fibroblast transdifferentiation model was verified by qRT-PCR.3.GO(Gene Ontology,GO)and KEGG(Kyoto Encyclopedia of Genes and Genomes,KEGG)enrichment analysis and prediction were performed for the verified differential circRNA Genes.4.Online library of mi Randa,Target Scan and RNAHybrid were used to predict the mi RNAs that might be targeted by different circRNAs,and then to predict the m RNAs that might be activated by mi RNAs.Cyctoscope was used to construct the screened and verified differentially expressed circRNAs ce RNA network.Results:1.Analysis of high-throughput sequencing results showed that compared with normal lung tissue,601 obviously abnormal circRNAs were identified in the lung tissue of patients with IPF,among which 237 were upregulated and 364 were down-regulated.2.Western Blot analysis showed that the expression of a-SMA protein in IMR-90 cells was significantly increased after 72 h treatment with TGF-?1(5ng/ml).The in vitro model was established successfully.3.Verification of hsa_circ₀001092 and hsa_circ₀002490 by qRT-PCRIn the TGF-? 1-induced lung fibroblast transdifferentiation model,the expression of hsa_circ₀026782 was low and the expression of hsa_circ₀005328was high,which was consistent with the sequencing results.However,the up-regulated hsa_circ₀005328 showed no significant difference in the verification results.The down-regulated hsa_circ₀006354 and hsa_circ₀000471 were contrary to the predicted results.4.GO and KEGG enrichment analysis was performed for the differentially expressed circRNAs.Function prediction GO find differentially expressed genes mainly with the development of projection neurons regulation,regulating cell morphogenesis,Ras protein signal transduction,regulation and control of cells(cell frontier,synaptic membrane,synaptic)glutamate can,DNA-the combination of transcriptional activation activity,RNA polymerase ?-specificity,sequence specific DNA binding,bacterial RNA polymerase Transcriptional process,etc.KEGG signaling pathway results showed that the differentially expressed circRNA in TGF-?1-induced lung fibroblast transdifferentiation model was mainly related to erb B signaling pathway,MAPK signaling pathway,Hippo signaling pathway,sphingolipid signaling pathway,signaling pathway regulating pluripotency of stem cells,adhesion plaques,and actin cytoskeleton regulation.5.Construction of ce RNA regulatory networks with differential circRNAs.Predicting differential circRNAs as possible downstream molecules.Conclusion:1.Analysis of high-throughput sequencing results showed that compared with normal lung tissue,601 obviously abnormal circRNAs were identified in the lung tissue of patients with IPF,among which 237 were upregulated and 364 were down-regulated.2.The expression of hsa_circ₀001092,hsa_circ₀002490 and hsa_circ₀026782 were verified,and GO and KEGG enrichment analysis was performed on the differential circRNAs.Idiopathic pulmonary fibrosis may be related to the Ras protein signal transduction,erb B signaling pathway,MAPK signaling pathway,HIPPO signaling pathway,actin cytoskeleton regulation,etc.It is suggested that these differentially expressed circRNAs may be involved in the occurrence and development of idiopathic pulmonary fibrosis through the regulation of multiple processes of idiopathic pulmonary fibrosis.3.The ce RNA regulatory network of circRNAs was established,suggesting that all the regulatory pathways involved in the screened genes could become new targets for the intervention treatment of idiopathic pulmonary fibrosis.