Study On Differential Expression And Functional Prediction Analysis Of Non-coding RNA In Rats With Hypoxic Pulmonary Hypertension

Objective:Hypoxic pulmonary hypertension(HPH)is closely related to the course and prognosis of chronic pulmonary heart diseases caused by chronic obstructive pulmonary disease and pulmonary fibrosis,and has brought a heavy burden on society and families.It has become a major disease that seriously endangers the health of the people.We intend to introduce the transcriptome and combine bioinformatics methods to explore non-coding RNAs expression characteristics in HPH rat model lung tissues,such as Circular RNA,(circRNA),long non-coding RNA(IncRNA),and microRNA(miRNA).And then screen differentially expressed genes,analyze key genes and construct a network of competing endogenous RNA(ceRNA),identify their target genes and functions relevant molecular mechanisms are designed to explore the physiological and pathological mechanisms of HPH from the perspective of ncRNA.The purpose of this research is to clarify the roles and mechanisms of their possible occurrence,development and prevention,and to provide reliable theoretical basis and therapeutic targets for the effective prevention and treatment of HPH.Methods:A HPH rat model was constructed.Pulmonary arterial pressure was measured using the right heart catheter method,right ventricular hypertrophy index was calculated,pathological sections of pulmonary small blood vessels were processed,and their reconstruction was analyzed.The above method was used to check whether the HPH rat model was successfully copied.The HPH group and the control group were randomly selected from three rat lung tissues for high-throughput sequencing of the whole transcriptome.Bioinformatics methods were used,including differential gene analysis,target gene prediction,GO function enrichment analysis,KEGG pathway enrichment analysis,and protein interactions analysis,etc.,which were to identify important genes and ceRNA regulatory networks involved in HPH disease.The partially differentially expressed ncRNAs and mRNAs were screened,and quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to verify the sequencing results.Results:1.Compared with the control group,hypoxia induction significantly increased right ventricular pressure(RVSP)in the HPH group(33.67±1.53 mmHg vs 17.33±2.08 mmHg,P<0.001),and right ventricular hypertrophy index(RVHI)also increased significantly(0.41±0.02 vs 0.28±0.01,P<0.001).Histopathological staining suggested that hypoxia could induce pulmonary arteriolar remodeling(80.17 ± 2.31%vs 59.37±3.74%,P<0.01).The results showed that the HPH rat model was successfully constructed.2.According to the standard P<0.05 and | log2FoldChange |>1,select 15 significant differential expressions circRNAs(Differentially Expressed circRNAs,DEcircRNAs)(11 up-regulated,4 down-regulated),61 significant DEIncRNAs(32 up-regulated,29 down-regulated),9 significant DEmiRNAs(7 up-regulated,2 down-regulated),212 significantly DEmRNAs(141 up-regulated,71 down-regulated).3.GO function and KEGG pathway enrichment analysis of DEmRNAs in sequencing results,the up-regulated mRNAs were mainly concentrated in chronic inflammatory response,response to oxidative stress,response to interleukins,NF-κB signaling,smooth muscle cell proliferation,smooth muscle contraction,autophagy,endothelial cell chemotaxis,and chemokines Mediated signaling pathway,Toll-like receptor signaling pathway,PI3K-Akt signaling pathway.HIF-1 signaling pathway and so on.The down-regulated mRNAs were mainly concentrated in immune response,smooth muscle cell differentiation,Toll-like receptor signaling pathway,etc.4.Analysis of DEmRNAs PPI in sequencing results found that up-regulated mRNAs in the PPI network,the core genes were Tlr2,Cxcr2,Mmp9,Ptafr,Mpo,Mmp8,Pglyrpl,Fpr1,Oscar,Defa5,Camp,Fos,Csf3r.Down-regulate mRNAs in the PPI network,the core genes were Rtp4,Irf7,Herc6,Oas2,Rsad2,Mx2,Mx1,Isg15,Usp18,Gbp4.5.The circRNA-miRNA-mRNA ceRNA regulatory network contained 6 DEcircRNAs(4 up-regulation,2 down-regulation),4 DEmiRNAs(2 up-regulation,2 down-regulation)and 22 DEmRNAs(9 up-regulation,13 down-regulation),a total of 51 pairs of circRNA-miRNA-mRNA ceRNA.Functional enrichment analysis of mRNAs in the network,the main functions were regulation of inflammatory response,regulation of immune response,cell response to redox state,arterial smooth muscle contraction,positive regulation of nitric oxide metabolism,positive regulation of Toll-like receptor signaling,regulation of transduction pathways,negative regulation of interleukin production,negative regulation of chemokine production,etc.6.The lncRNA-miRNA-mRNA ceRNA regulatory network contained 43 DElncRNAs(20 up-regulated,23 down-regulations),9 DEmiRNAs(7 up-regulations,2 down-regulations)and 27 DEmRNAs(9 up-regulations,18 down-regulations),a total of 450 pairs of lncRNA-miRNA-mRNA ceRNAs.Functional enrichment analysis of mRNAs in the network,the main functions were regulation of immune response,cell response to redox state,contraction of arterial smooth muscle,regulation of Toll-like receptor signal transduction pathway,negative regulation of interleukin production,chemotaxis negative regulation of factor production.7.The results of qRT-PCR verification were consistent with the results of sequencing.Conclusion:1.The HPH rat model was successfully constructed.2.The transcriptome genes(circRNAs,lncRNAs,miRNAs,mRNAs)were differentially expressed in the lung tissue of normal control and HPH rats,which suggested that they may be used as potential biomarkers for HPH screening.3.Many core gene mRNAs were involved in HPH-related biological functions and signaling pathways.4.circRNAs and IncRNAs can combine multiple HPH-related miRNAs to exert ceRNA regulation mechanism.