Bioinformatics Of Gene Expression And Mechanism Of Key Molecule CUL2 In Pulmonary Arterial Hypertension

Background:Pulmonary arterial hypertension(PAH)is a group of diseases with increased pulmonary vascular pressure.The pathogenesis of PAH is clearly related to multiple gene mutations.With the development of high-throughput genomics technology,PAH samples can be sequenced quickly to detect the variation of any part of the genome.This research method can explore the pathogenesis of PAH efficiently and completely at the gene level.The key initial of PAH is endothelial injury.Hypoxia can stimulate the proliferation of pulmonary artery endothelial cells(PAECs).Previous studies have confirmed that PAECs are related to ubiquitination.Bioinformatics analysis showed that after gene differential expression and pathway analysis,the structural proteins CUL1 and CUL2 of E3 ubiquitin ligase were significantly up-regulated in PAH samples.However,the interaction between CUL1,CUL2 and PAECs is unknown,and the regulatory mechanism of CUL1 and CUL2 on PAECs is still unclear.Objective:This study would identify that: 1.to screen the possible differential expression genes in PAH disease,2.to explore the expression changes of differentially expressed genes at the cellular level to verify the results of bioinformatics,3.hypoxia intervention in PAECs to clarify the possible regulatory mechanism of the differentially expressed genes in PAH.Methods:1.The microarray data of PAH patients were extracted from the public database for gene function and pathway enrichment analyses.2.Construct differentially expressed genes and pathways,and construct proteinprotein interaction network.3.Partial screened genes(CUL1,CUL2,CUL3,CDC5 L,DHX15,EIF2AK2,TOPBP1,ACE,DLL4 and EGFL7)were extracted for differential expression verification.4.PAECs were extracted and cultured,and the expression levels of CUL1 and CUL2 were detected under hypoxia stimulation.5.To detect the effect of hypoxia on the proliferation and migration of PAECs.6.After transfection of PAECs with CUL2 interfering adenovirus vector,the protein expression level and the proliferation and migration ability of PAECs were detected under hypoxia stimulation.Results:1.Bioinformatics analysis showed that there were 1442 common differentially expressed genes in Congenital Heart Disease associated PAH(CHD-PAH),Connective Tissue Disease associated PAH(CTD-PAH)and idiopathic PAH(IPAH),among which 1158 were up-regulated genes,accounting for 80.31%.2.Gene Ontology(GO)functional analysis indicated that the up-regulated genes were mainly composed of intracellular and organelle components,and "binding" function was involved in the metabolic process including cell cycle.The down regulated genes are mainly composed of cell membrane components,which are involved in the function of cytochrome c oxidation activity and negative regulation of migration and movement of cell components.3.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis indicated that the signal pathways involved in cell cycle and DNA damage were enriched.4.Protein-protein interaction(PPI)network and modules indicated that genes encoding cell cycle and DNA damage related proteins are highly interacting genes.As new candidate genes related to PAH,these genes are worthy of further study.5.The verification of seven differentially expressed genes showed that the expression of CUL1,CUL2,CUL3,CDC5 L,DHX15,EIF2AK2,TOPBP1,ACE,DLL4 and EGFL7 was significantly dysregulated under hypoxia stimulation.6.Both differentially expressed genes and highly interacting genes were found,CUL1 and CUL2 not only have up-regulated differential expression in PAH,but also participate in both cell cycle and ubiquitination.CUL1 is an important interaction gene(hub)in PPI network.7.Under hypoxia stimulation,CUL2 protein expression of PAECs increased significantly,but CUL1 protein expression had no significant difference.The proliferation and migration of PAECs were also significantly enhanced.8.Under hypoxic stimulation,the overexpression and migration of PAECs were inhibited by down regulating the expression of CUL2.Conclusion:1.Bioinformatics analysis showed that there were common differentially expressed genes among CHD-PAH,CTD-PAH and IPAH,and 80.31% of them were up-regulated.Go function analysis,KEGG pathway enrichment analysis and PPI network diagram showed that genes and signaling pathways related to cell cycle and DNA damage were significantly enriched.2.The differentially expressed genes(CUL1,CUL2,CUL3,CDC5 L,DHX15,EIF2AK2,TOPBP1,ACE,DLL4 and EGFL7)were dysregulated in hypoxia,which verified the bioinformatics results.3.Under hypoxia stimulation,CUL2 affects the biological behavior of endothelial cells by regulating the proliferation and migration of PAECs.