Bioinformatics Analysis For Specific Genes Of Sepsis,Biomarker Screening And Studies On The Function Of ANXA3 In Sepsis

Sepsis is a life-threatening multi-organ dysfunction caused by the dysregulated host response to infection.Sepsis is one of the serious complications in patients with severe infections,and can eventually lead to multiple organ failure or fatal results of patients in the intensive care units(ICU).However,the pathogenesis of sepsis is not very clear yet,and there still lacks specific biomarkers and effective therapeutic targets.Bioinformatics is an important tool in searching for pathogenesis,biomarkers and therapeutic targets of diseases.In recent decades,the widespread use of genome-wide technologies such as DNA microarrays and RNA high-throughput sequencing has made it possible to rapidly assess the genome expression profiles.The massive amount of data generated by genome-wide analysis has facilitated the generation of new models and algorithms in bioinformatics,which have played an important role in the research of mechanisms,biomarkers and therapeutic targets in diseases such as inflammation,cardiovascular diseases and cancer.This research started from bioinformatics to study the pathogenesis,biomarkers and key genes of sepsis.In the first part of this study,the key genes,pathways,proteins,transcription factors and mi RNAs in sepsis were searched.First,we analyzed multiple datasets around the world,and screened out the differentially expressed genes(DEGs)shared in the whole blood of sepsis patients of different races.Then,by using databases such as Gene Oncology(GO),Reactome,KEGG Pathway,PANTHER,and Bio Cyc,we revealed the biological processes,cellular components,molecular functions,and pathways that these genes significantly enriched in.Next,this part of the study utilized the STRING database to construct a Protein-Protein Interaction Network(PPI Network)for the proteins corresponding to the differentially expressed genes,and used the Cytoscape software to search for key node proteins and areas with more protein interactions.Next,the transcription factor-gene interaction networks were constructed using the ENCODE database to find the key transcription factors in sepsis.Then,10 databases including mi Records,mi RTar Base,Tar Base,mi R2 Disease,HMDD,Phenomi R,SM2 mi R,star Base,Pharmaco-mi R,and Epimi R were used to construct mi RNA-gene interaction networks to screen mi RNAs that may play a therapeutic role.Finally,we used the Weighted Gene Correlation Network Analysis(WGCNA)algorithm to screen for genes most related with clinical diagnosis through the removal of outliers,the construction of scale-free networks,gene clustering and module construction.The results in this part showed that 444 genes were significantly differentially expressed in the whole blood of sepsis patients across different regions and races,including 246 up-regulated genes such as MMP8 and CD177,and198 down-regulated genes such as SBK1 and PRSS33.These genes were mainly enriched in biological processes such as "innate immune response",cellular components such as "T cell receptor complex",molecular functions such as "MHC class II protein receptor activity",and pathways such as "neutrophil dysregulation".In the protein-protein interaction(PPI)network,we found key node proteins such as ITGAM,LCK,TLR8,CD28,CCL5,CCR7,MMP9,CD2,ZAP70,CD4,CD3 E.These proteins are often at the core of pathogenesis due to more interactions,and they are also more likely to act as targets for drug intervention.In addition,2areas with dense interactions were also found in the PPI network,and they are related to pathways such as "neutrophil degranulation" and "immune system".Notably,Arginase 1(ARG1)protein is centrally located in the first area,suggesting it is more relevant to the pathogenesis of sepsis.Next,this part of the study revealed the key transcription factors such as TFDP1 and IRF1 that act on up-regulated genes,and key transcription factors such as WRNIP and HDGF that act on down-regulated genes.Given that many drugs target transcription factors,it is of great value to reveal the crucial transcription factors related to sepsis.In addition,this study also identified potentially therapeutic mi RNAs such as hsa-mir-218-5p,hsa-mir-193b-3p that specifically target upregulated DEGs,and mi RNAs such as hsamir-192-5p and hsa-let-7b-5p that specifically target downregulated DEGs.The application of these mi RNAs or their antisense nucleic acids may help to maintain the homeostasis of gene expression during sepsis.Last,the expression profile data of differential genes were modularized by WGCNA analysis,and a total of 15 genes most closely related to the clinical diagnosis of sepsis,including Annexin A3(ANXA3),were discovered.Notably,among the key node proteins,transcription factors,mi RNAs,and genes most closely related to clinical diagnosis discovered in this part,many have been reported to have clear associations with sepsis,which confirmed the reliability of our research methods.In conclusion,in this part,multiple bioinformatics approaches were integrated to systematically and comprehensively screened the key genes,proteins,transcription factors,and mi RNAs associated with sepsis.These findings could contribute to the understanding of the pathogenesis of sepsis and provide biomarkers and potential therapeutic targets for the diagnosis and treatment of this disease.In the second part,we focused on the potential of the two genes,Arginase 1(ARG1)and Annexin A3(ANXA3),as biomarkers,and further explored their mechanisms.First,we found that ARG1 was the only gene shared by the results of PPI network screening and WGCNA algorithm.Also,ARG1 was located at the core position of the first area with dense interactions in the PPI network.This indicated that ARG1 was more closely related to sepsis and shows more potential as a biomarker.Next,this study used 10 datasets from different regions(five from North American,four from European,one from Asian),ages(six from adults,four from children)and technical methods(eight from DNA microarrays,two from RNA-sequencing).It was found that the transcript abundance of ARG1 was significantly increased in the peripheral blood of sepsis patients compared to healthy controls or control patients,regardless of ethnicity,age or experimental technique.Then,by using Receiver Operating Characteristic(ROC)curves,it was demonstrated that the ARG1 gene can achieve high sensitivity while maintaining high specificity,fully demonstrating its potential as a biomarker.Moreover,a good biomarker should not only effectively distinguish patients from healthy controls,but also should play a role in accurate diagnosis,judgment of severity,and prognosis prediction.Therefore,we next examine the performance of ARG1 at other levels.Our study found that the expression levels of ARG1 were significantly higher in whole blood of patients with septic shock than in non-septic shock.Given that both types of shock share similar symptoms,ARG1 is valuable as a biomarker in clinical practice for the specific diagnosis.Moreover,this study found that ARG1 was significantly up-regulated in whole blood of patients with severe sepsis and fatal death than in patients with general sepsis,suggesting that ARG1 could playing a role in predicting the severity of sepsis.Next,we found significantly higher expression of ARG1 in sepsis patients who did not respond to subsequent supportive therapy,suggesting ARG1 could help to predict the outcome of treated sepsis patients.We also performed sepsis modeling at the animal level with Cecum Ligation and Puncture(CLP),and confirmed high expression of ARG1 in whole blood of septic mice.We also used GSEA analysis to explore the mechanisms of ARG1’s up-regulation,and the results showed that it was associated with starch and sucrose metabolism,sphingolipid metabolism,and ferroptosis.As for the ANXA3 gene,this study also researched the potential as a biomarker for sepsis since the WGCNA results showed a significant association between this gene and the clinical diagnosis.Similar to ARG1,ANXA3 can effectively differentiate sepsis patients from healthy controls.However,ANXA3 only showed up-regulation in whole blood of septic shock patients than in cardiogenic shock patients,and cannot effectively predict the severity of sepsis and whether there is a response to supportive treatment.After animal experiments confirmed the high ANXA3 expression during sepsis,mechanistic exploration also suggested that high ANXA3 expression in the blood is associated with the SNARE complex.In conclusion,the second part researched the value of ARG1 and ANXA3 as biomarkers for sepsis diagnosis,prognosis,and response to treatment.Given that many sepsis biomarkers are derived from inflammatory responses,lack of specificity for sepsis,and only have single function,ARG1 could act as a specific and multifunctional biomarker to remedy this shortcoming.The third part of this study focused on the role of ANXA3 in sepsis.Among the top 5genes most closely associated with sepsis diagnosis identified by WGCNA in the previous part,four genes have already shown a clear correlation or even a "causal" association with sepsis,while only the association of ANXA3 has not been reported experimentally.Therefore,we decided to explore the function of ANXA3 gene in sepsis.In this part,we first confirmed the high expression of ANXA3 at the protein level in whole blood of septic mice by Western Blot,and then used immunohistochemistry and found that ANXA3 protein expression was significantly higher in the spleen and lower in the lung vascular endothelium in septic mice compared to Sham groups.Also,this protein rarely expressed in the liver,and did not show significant changes after sepsis modeling.Next,by using flow cytometry,we showed that ANXA3 protein expression was significantly upregulated on macrophages,neutrophils,total myeloid cells,and B cells in septic mice,while do not express on T cells before or after sepsis.Next,we constructed ANXA3 knockout mice and verified the genotype from three levels:DNA,m RNA and protein.Finally,the potential roles of ANXA3 gene in sepsis were explored in terms of organ function,tissue damage,bacterial load,cellular infiltration,and survival using cecal ligation and puncture(CLP)and intraperitoneal lipopolysaccharide(LPS)models respectively.The results showed that there were no significant differences between wild-type septic mice and ANXA3 knockout septic mice in these aspects,which may be related to the "beneficial" effect of ANXA3 in clearing bacteria and repairing tissue damage,offset by "detrimental" function of prolonging neutrophil lifespan and increasing inflammatory response.In conclusion,first,this study integrated multiple bioinformatics analysis methods and data,and uncovered multiple genes,proteins,transcription factors and mi RNAs closely related to sepsis.Second,this study identified two potential biomarkers of sepsis,ARG1 and ANXA3,and examined their performance in accurate diagnosis,prognosis prediction,and prediction of response to treatment.Third,through the construction of ANXA3 gene knockout mice,this study further explored the role of ANXA3 gene in organ function,tissue damage,bacterial load,cell infiltration and survival in CLP and LPS-induced sepsis.These findings can deepen the understanding of the molecular mechanisms involved in the development and progression of sepsis,and contribute to the accurate diagnosis and treatments.