Identification Of Key Pathways And Genes In Carotid Atherosclerosis Through RNA-seq Data And Explore The Effect And Mechanism Of SAA1 On Macrophages

Part ? Identification of key pathways and genes in carotid atherosclerosis through bioinformatics analysisBackgroundAtherosclerosis(AS)is the main pathological basis of myocardial infarction,ischemic stroke,and peripheral vascular disease,,and its development is by chronic inflammation and autoimmune involved in many factors,multi-stage development of complex events,prevent atherosclerosis at present mainly concentrated in reduce inflammation and reduce blood fat and surgery.How to make early diagnosis and treatment intervention to prevent the progression of atherosclerosis has become the main strategy of prevention and treatment of the disease.Therefore,it is urgent to further explore the mechanism of atherosclerosis occurrence and development and find effective prevention and treatment targets in the early stage.Transcriptome sequencing(RNA-SEQ)is a powerful tool for gene exploration to elucidate the pathogenesis of disease.In this study,we performed RNA-SEQ analysis of arterial intima tissue from patients with carotid atherosclerosis(CAS)and healthy individuals.Differentially expressed genes(DEGS)associated with CAS were screened out,and various functional analyses(including GO,KEGG and MCODE enrichment analysis)were performed on DEGS.The results showed that DEG was mainly concentrated in biological pathways related to inflammation and immunity.Twenty Hub genes were screened by constructing a protein interaction network(PPI).Bioinformatics analysis showed that Hub genes associated with AS were mainly concentrated in the lung,and their core transcription factors were Rela and NFKB1,which were involved in a variety of biological processes and disease development.CMAP database identified three small molecule compounds that interact with Hub genes:Ceforanide,Chenodeoxycholic Acid and 0317956-0000,which can target and regulate THE Hub genes associated with AS,and are expected to be targeted drugs for the treatment of AS.In this study,RNA-SEq was combined with bioinformatics to explore the pathogenesis and potential treatment of AS.ObjectiveIn this study,RNA-SEQ sequencing was performed on carotid atherosclerosis and normal intima tissues to analyze the differential gene expression profile.Combined with bioinformatics analysis,key pathways and genes related to atherosclerosis were screened to explore the potential mechanism of atherosclerosis and potential therapeutic targets.Methods1.The intima of carotid atherosclerosis patients and the arteries of previously healthy trauma patients were collected and tested for RNA sequencing(RNA-SEQ).RNA was extracted by TRIzol method,and Illumina sequencing was performed after library construction and quality inspection.Data quality control,the original data were filtered,and the clean reads after quality control were compared to the human reference genome using HISAT2 software to obtain satisfactory sequencing data.The gene expression distribution and quantitative analysis met the expected requirements.2.GO and KEGG pathway analysis of differential genes.We first converted the gene ID from the Official Symbol of the differential gene obtained by org.Hs.eg.Db package and clusterProfiler package were adopted for GO and KEGG pathway analysis,respectively.Besides,the GOplot package was used for cluster analysis.Adj.p.Val<0.05 denoted statistical significance.3.MCODE analysis:Differentially expressed genes(DEGs)were analyzed using Metascape online database.KEGG pathway enrichment analysis was performed for each MCODE.p<0.01 denoted statistical significance.4.PPI network construction and hub gene screening:STRING database was used to construct a PPI network.An interaction with a composite score greater than 0.9 was considered statistically significant.CytoHubba was used to evaluate PPI network hub genes.The top 20 hub genes were screened out through CytoHubba Plugin by degree..5.Bioinformatics analysis of hub gen:The top 20 hub Genes were analyzed via Metascape online database.Interaction of small-molecule drugs with hub genes was predicted using CMAP online database.Results1.The Basic analysis of sequencing dataAfter filtering the original data,checking the sequencing error rate,and verifying the distribution of GC content,the clean reads for follow-up analysis were obtained.Carotid atherosclerosis samples ASA₁ and ASA₂ generated 57.14,and 72.76 million clean reads,respectively.Normal control samples NA₁,NA₂,and NA₃ generated 59.84,70.13,and 59.98 million clean reads,respectively.Quality-controlled clean reads were compared to the human reference genome.Results of the comparison of each sample in this project with the human reference genome are presented in Table 2.Total mapped reads for ASA₁ and ASA₂ were 96.08 and 94.70%,whereas those for NA₁,NA₂,and NA₃ were 94.95,96.28,and 95.99%,respectively.2.Gene expression analysis and Identification of differentially expressed genesBox and density diagrams depicted uniform distribution of gene expression levels in different samples.Based on correlation analysis of gene expression levels among samples,the experiment was reliable with accurate sample selection.Principal component analysis(PCA)revealed that samples were scattered among groups and clustered within groups.After normalizing the sequencing data,we constructed the heat map.A total of 1427 DEGs were identified after screening at |logFC|>3 and adj.p.Val<0.05;710 DEGs and,717 DEGs were up-regulated and down-regulated,respectively.3.GO and KEGG enrichment analysis of DEGsGO analysis revealed that biological processes(BP)of these targets were mainly enriched in leukocyte migration,regulation of activation,and inflammatory response.Enriched cell components(CC)included extracellular matrix,plasma membrane protein complex,and synaptic membrane.Molecular functions(MF)were mainly enriched in Metal ion Transmembrane Transporter activity,Substrate-specific channel activity,and amide binding.KEGG pathway analysis demonstrated that enriched pathways for these targets are implicated in neuroactive ligand-receptor interaction,cytokine-cytokine receptor interaction,and cell inflammatory molecules.Cluster analysis for the top five GO terms explored the associations of genes such as MMP12,MMP8,and MMP9 with phagocytosis,leukocyte migration,acute response,regulation of immune activation,and regulation of immune effector process.We performed cluster analysis for the top 5 KEGG pathways to explore the association of genes such as LBP,CSF3,and FCGR2A with leishmaniasis,hematopoietic cell,strain,tuberculosis,and osteoclast differentiation.4.Enrichment analysis of DEGs in MCODEMCODE analysis demonstrated that DEGs were grouped into 18 modules.Briefly,Module 1 was comprised of 29 genes,including GAL,GNAl1,and OPRK1,etc.Module 2 comprised 11 genes,including TACR1,NTSR1,and TAC1 etc.Module 3 comprised 11 genes including CD74,CTSS,and CTSV,etc.We did KEGG enrichment analysis on the 18 modules.Pathways in module 1 were mainly associated with neuroactive ligand-receptor interaction,whereas those in module 2 and module 3 were mainly enriched in cAMP signaling and chemokine signaling,respectively,and so on.5.Constructing PPI network and screening for hub genesThe STRING online Database was employed to construct the PPI network of DEGs,which was then visualized and optimized using Cytoscape software.Analysis of hub genes was achieved using the degree method in cytoHubba;the top 20 genes were identified as hub genes.6.Bioinformatics analysis of hub genesEnrichment analysis of top 20 hub genes in the Metascape database revealed that these genes are mainly associated with Galpha(i)signaling events,GPCR ligand binding,and neuroactive ligand-receptor interaction.TRRUST database analysis uncovered RELA and NF-?B1 to be the main transcription factors regulating the hub genes.DisGeNET database disease enrichment analysis demonstrated that the hub genes were associated with depressive disorder,mental depression,and alcoholic intoxication.Besides,PaGenBase database tissue characteristic enrichment analysis showed that hub genes were mainly enriched in the lung.Analysis of the CMAP database showed that CeForanide,Chenodeoxycholic acid,and 0317956-0000 compounds potentially exerted a negative regulatory effect on hub genes.We deduced that these three drugs are potential candidates for the management of carotid atherosclerosis.Conclusions1.In the present study,we employed the R package(DESeq2)to analyze the sequencing data set and yielded 1,427 DEGs(710 up-regulated and 717 down-regulated).This finding demonstrates that carotid atherosclerosis is associated with multiple gene regulatory changes.2.GO and KEGG enrichment analysis revealed that expression changes of these DEGs influenced signal transduction pathways associated with inflammation and immune response,for example,cytokine-mediated signal transduction and inflammatory response.3.Function-based grouping of the DEGs into 18 modules affirmed their different effects in the pathological process of CAS.Also,the regulation of module genes is interrelated.In a nutshell,the pathological mechanism of CAS is a complex process,associated with ligand-receptor interaction of the nervous system,cAMP signaling pathways,chemokine signaling pathways,and other biological processes.4.Using the CMAP database,we identified three small molecular compounds(CeForanide,Chenodeoxycholic acid,and 0317956-0000)that potentially interact with central genes.The small molecule drugs that we screened may exert potential activity against atherosclerosis.SignificanceIn conclusion,the present study adopted RNA-seq technology and bioinformatics analysis to explore the pathogenesis of CAS.It demonstrates that inflammation,immune response,and neuroligand-receptor interaction are associated with the development of CAS.Also,20 hub genes and 3 candidate small-molecule drugs for CAS treatment.Part ? To explore the effect and mechanism of SAA1 on macrophage THP-1 through p38MAPK signaling pathwayBackgroundThe formation of atherosclerosis is a complex multi-factor,multi-step and multi-stage event involving oxidative stress,inflammatory cell aggregation,endothelial dysfunction,low density lipoprotein(LDL)oxidation,foam cell formation and vascular smooth muscle cell proliferation and migration.On research to the pathogenesis of AS for hundreds of years of history,the main theory of doctrine of endothelial injury reaction and lipid infiltration theory,theory of thrombosis,homocysteine theory,theory of smooth muscle cell cloning,shear stress theory,immune theory and so on,its specific pathogenesis is not fully proved,still to be further study for sure.With the development of molecular biology and gene diagnosis,it has become a hot topic to elucidate the pathogenesis of atherosclerosis from molecular and gene level.Therefore,there is a growing need to identify innovative therapeutic targets and new antiatherosclerotic agents.In the previous study,rnA-SEQ analysis was performed on intima tissues of CAS patients and healthy subjects by transcriptome sequencing technology(RNA-SEQ).Combined with biological information analysis,genes expressed significantly differently in atherosclerosis patients were screened compared with healthy subjects.Twenty Hub genes related to atherosclerosis were further identified,among which SAA1,GNG8 and LPAR2 had the highest levels(with the strongest correlation).We treated thP-1-derived macrophages with low-density oxidized lipoprotein(OX-LDL)and detected the expression of SAA1,GNG8 and LPAR2 normal THP-1 macrophages and OX-LDL-treated THP-1 macrophages by real-time fluorescence quantitative polymerase chain reaction(QRT-PCR).The most differentially expressed SAA1 was selected for follow-up study.SAA1 mainly encodes human serum amyloid A at the acute stage.A-saa is an acute reactive protein,which can be expressed by SAA1 when the body is stimulated by inflammation or immunity,and then synthesized in large quantities in the liver and adipose tissue and secreted into the blood.A-saa has A variety of biological functions,which can promote the adhesion,migration and infiltration of inflammatory cells,inhibit immune response,influence cholesterol metabolism by combining with high-density lipoprotein,and induce the expression of multiple matrix metalloproteinases.A-saa is widely involved in the regulation of inflammation,immunity,lipid metabolism,cardiovascular and cerebrovascular diseases,tumors and other diseases.We intend to study the effect of SAA1 on the inflammatory response and oxidative stress of macrophages,an important participant in the occurrence of AS,and explore its possible mechanism through cell experiments,in order to provide a new target for the prevention and treatment of atherosclerosis.Objective1.Thp-1 macrophage model of atherosclerosis was constructed by incubating THP-1 with OX-LDL.2.Verify the expression levels of Hub genes screened out by RNA-SEQ sequencing,and conduct further research on those with the most obvious expression differences.3.The selected Hub genes were interfered and overexpressed,respectively,to study their effects on the expression of inflammatory cytokines(TNFa,IL6,iNOS)and cytokines involved in oxidative stress(ROS,MDA,SOD)in THP-1 macrophage model.4.Explore the possible mechanism of Hub gene affecting THP-1 macrophages by detecting the expression of pathway proteins.5.This study aims to explore the effect and mechanism of Hub gene SAA1 on THP-1 macrophages and explore the role and mechanism of SAA1 in the occurrence and development of atherosclerosis,so as to provide a new therapeutic target for the prevention and treatment of atherosclerosis.Methods1.Construction of atherosclerotic THP-1 cell environmentIn the experimental group,THP-1 was incubated with OX-LDL to construct atherosclerotic cell model,and the growth environment of AS cells was constructed by adding OX-LDL.Thp-1 without special treatment was used as control group.We used ox-ldl at concentrations of 0,6.25,12.5,25,50,100 and 150 ug/mL to intervene macrophages(thp-1)and cck-8 cell proliferation assay to detect the effects of different ox-ldl concentrations on thp-1 cell proliferation.2.Validation of Hub gene expression in THP-1 cell modelThe expression differences of three Hub genes,SAA1,GNG8 and LPAR2,which are most correlated with atherosclerosis,were detected by RT-qPCR between the experimental group and the control group of THP-1,and the most significant differential expressions were selected for follow-up study.3.Changes of inflammatory factors and cytokines were detected after hub gene transfectionThp-1 macrophages were transfected with SAA1-mimic and inhibitor respectively,and the changes of AS related inflammatory factors and cytokines such AS TNFa,IL6,iNOS,ROS,MDA and SOD in THP-1 cells after transfection with SAA1-mimic and inhibitor were detected by ELISA.4.Changes of pathway proteins after transfection of HUB gene,and the possible mechanism was studiedThp-1 macrophages were transfected with SAA1-mimic and inhibitor,respectively,and the changes of P38 mitogen-activated protein kinase(P38 MAPK)and its activated phosphorylated form p38MAPK(P-P38 MAPK)protein in the interference group and the normal group were detected by Western blot.5.Changes of inflammatory factors and cytokines were detected after the addition of pathway protein inhibitors to further verify the mechanismSB203580,an inhibitor of P38 MAPK signaling pathway,was added into THE transfection of SAA1.Enzyme linked immunosorbent assay(ELISA)was used to detect the changes of AS related inflammatory factors and cytokines,including TNFa,IL6,iNOS,ROS,MDA and SOD in THP-1 cells after transfection of SAA1.Results1.Thp-1 was incubated with OX-LDL to construct atherosclerotic cell modelThe experiment indicated that OX-LDL could effectively inhibit the proliferation of THP-1 cells.When ox-LDL concentration reached 50 ug/mL,thP-1 cell proliferation could be significantly inhibited(the inhibition rate was 60.5%,and the formation of foam cells was satisfactory under the microscope).The intervention concentration of Ox-LDL was 50 ug/mL.The intervention time was 24 hours.2.Expression level verification--THE expression levels of SAA1,GNG8 and LPAR2 in the experimental group and control group were detected by RT-PCRThe expression of three Hub genes SAA1,GNG8 and LPAR2 were detected by RT-QPCR between thP-1 model group and normal control group.Results The expression ratio of SAA1,GNG8 and LPAR2 in thP-1 model group and normal control group was 3.69(P<0.01),2.15(P<0.01),1.95(P<0.01),in this study,it could be seen that the expression of three Hub genes,SAA1,GNG8 and LPAR2,were significantly up-regulated in THP-1 cells incubated with 50 ug/mL OX-LDL,among which SAA1 was the most significantly differentially expressed,so SAA1 was selected for follow-up study.3.Enzyme-linked immunosorbent assay(ELISA)was used to detect inflammatory factors and cytokinesThp-1 cells were transfected with SAA1-mimic and inhibitor,and the changes of AS related inflammatory factors and cytokines such AS TNFa,IL6,iNOS,ROS,MDA and SOD in THP-1 cells after transfection with SAA1-mimic and inhibitor were detected by enzyme linked immunosorbent assay(ELISA).The results showed that SAA1 could promote the release of inflammatory cytokines(TNFa,IL6,iNOS)and the expression of cytokines involved in oxidative stress(ROS,MDA)in OX-LDL-incubated THP-1 macrophages,and inhibit the expression of SOD,a cytokine with antioxidant effect.These results suggest that SAA1 may promote atherosclerosis by stimulating inflammatory response and oxidative stress.4.Western blot was used to detect protein changes in pathwayWestern blot was used to detect the changes of p38 mitogen-activated protein kinase(P38 MAPK)and its activated form phosphorylated p38MAPK(P-P38 MAPK)protein in the interference group and the normal group.Compared with normal group,p-P38 protein expression in model group was significantly up-regulated(P<0.005);Compared with model group,p-P38 protein expression in SAA1 mimic(overexpression group)was significantly up-regulated(P<0.005),while p-P38 protein expression was significantly down-regulated in si-SAA1 group(P<0.005).The expression of P-P38 protein in SAA1 overexpression group was significantly increased,suggesting that SAA1 can promote the inflammatory response and oxidative stress in atherosclerosis by activating the p38MAPK signaling pathway.5.Changes of inflammatory factors and cytokines were detected after p38MAPK inhibitionSB203580,an inhibitor of P38 MAPK signaling pathway,was added into THE transfection of SAA1.Enzyme linked immunosorbent assay(ELISA)was used to detect the changes of AS related inflammatory factors and cytokines,including TNFa,IL6,iNOS,ROS,MDA and SOD in THP-1 cells after transfection of SAA1.The results showed that SAA1 could promote the release of inflammatory cytokines(TNFa,IL6,iNOS)and the expression of cytokines involved in oxidative stress(ROS,MDA)in OX-LDL-incubated THP-1 macrophages,and inhibit the expression of SOD,a cytokine with antioxidant effect.SB203580 could significantly inhibit the above effects of SAA1(P<0.005),suggesting that SB203580,an inhibitor of P38 MAPK signaling pathway,can reverse the effects of SAA1-mediated inflammatory response and oxidative stress.Conclusions1.Through RNA-SEQ sequencing,the screened Hub gene SAA1 was significantly increased in thP-1 atherosclerotic cell model incubated with OX-LDL.2.SAA1 may promote atherosclerosis by stimulating thP-1 inflammation and oxidative stress.3.The effect of SAA1 on THP-1 macrophages is accomplished through the P38 MAPK signaling pathway,and the inhibitor SB203580 of P38 MAPK signaling pathway can effectively inhibit the inflammatory response and oxidative stress induced by SAA1 on THP-1.SignificanceThis study confirmed that SAA1 can promote the expression of inflammatory cytokines(TNFa,IL6,iNOS)and cytokines involved in oxidative stress(ROS,MDA)in THP-1 macrophages,and inhibit the expression of SOD,a cytokine with antioxidant effect.These results suggest that SAA1 may promote atherosclerosis by stimulating inflammatory response and oxidative stress.Mechanism studies showed that the effect of SAA1 on THP-1 macrophages was completed through the P38 MAPK signaling pathway,and the inhibitor SB203580 of P38 MAPK signaling pathway could effectively inhibit the above effects of SAA1.Inhibition of SAA1 is expected to be a new therapeutic target for the prevention and treatment of atherosclerosis.