| Part I Assciation of SYTL3 and SLC22A3 Polymorphisms with Serum Lipid Levels in the Guangxi Maonan and Han PopulationsBackground: Hyperlipidemia acts as one of the important risk factors of atherosclerotic cardiovascular disease(ASCVD).Previous studies have shown that abnormal lipid metabolism such as the elevated triglycerides(TG),total cholesterol(TC),apolipoprotein(Apo)B and low density lipoprotein cholesterol(LDL-C)levels and / or the decreased Apo A1 levels and high density lipoprotein cholesterol(HDL-C)are highly related to the occurrence and development of ASCVD.Hyperlipidemia is essentially a complex disease affected by genetic factors,environmental factors and their interactions.Previous studies have shown that the distribution of minimum allele frequency and genotype frequency of the same genetic variation locus and their relationship with blood lipid levels are different among different populations and races.Therefore,exploring the blood lipid level of a specific population,illustrating the influence of certain genetic variation factors on the blood lipid levels,and controlling it are of great significance for the prevention and treatment of cardiovascular and cerebrovascular diseases in a specific population,and even for guiding the lipidlowering treatment of the whole population.In recent years,a number of largescale genome-wide Association studies(GWASes)have uncovered some novel genetic loci that may be correlated with serum lipid levels,including synaptotagmin like 3(SYTL3)and solute carrier family 22 member 3(SLC22A3).Later in other populations,it is pointed out that several single nucleotide polymorphisms(SNPs)located in SYTL3-SLC22A3 gene cluster were associated with blood lipid levels in European people.Nevertheless,the correlation between serum lipid profiles and SYTL3-SLC22A3 SNPs has yet to be completely documented;the correlation of the new alternates and their haplotypes with the possibility of HLP in the Han or Maonan ethnic group has never been reported previously.Objective: To elucidate the distribution of serum lipid levels and their differences between Maonan and Han populations,and to explore the relationship between SYTL3-SLC22A3 SNPs and several environmental factors and serum lipid levels in two different ethnic populations.Methods: Total of 1436 unrelated people(668 males,46.52%;768 females,53.48%;749 Normal,52.16%;687 HLP,47.84%)of the ethnic Han group and 1393 unrelated individuals(624 males,44.80%;769 females,55.20%;631 Normal,45.30%;762 HLP,54.70%)of the ethnic Maonan group were selected using on previously stratified randomized cohorts.Epidemiological investigations are carried out in accordance with international standard methods.Stratified random sampling was conducted according to age and gender,and three generations of ancestors belonged to the Han or Maonan nationalities selected as the research subjects.In addition,all of the subjects were also confirmed by Y chromosome and mitochondrial diversity studies.The subjects in this study were all adults over 18 years old.Through questionnaire survey and physical examination,their eating habits,exercise style,smoking,alcohol consumption,height,weight,waist circumference,systolic blood pressure,diastolic blood pressure,BMI and pulse pressure difference were recorded.Total of 5 ml fasting venous blood sample was collected from each participant.A portion of the blood sample(2 ml)was collected in a test tube and used to measure blood lipid levels.The rest of the sample(3ml)was collected into a test tube containing anticoagulants(13.20 g/L tri-sodium citrate,14.70 g/L glucose,4.80 g/L citric acid)and utilized to extract deoxyribonucleic acid(DNA).Genotypes distribution amongst SYTL3-SLC22A3 SNPs in 2829 individual patients bearing no relationship to each other(Han,1436;Maonan,1393)were analyzed utilizing next-generation sequencing techniques.The distribution of allele frequency and genotype frequency of SYTL3-SLC22A3 SNPs in two different nationalities was further analyzed.Relationships between genotypes,haplotypes,environmental factors and their interactions and serum lipid levels or hyperlipidemia were evaluated in two ethnic groups.Results: 1.There are differences in general conditions and serum lipid levels between Maonan and Han population.The Maonan population possessed higher levels of LDL-C,diastolic blood pressure,Apo B,systolic blood pressure,TC,TG,weight,alcohol consumption,pulse pressure and BMI in contrast to the Han group(P < 0.05-0.001).However,levels of serum Apo A1,the Apo A1/Apo B ratio and HDL-C were decreased in those of Maonan ethnicity in contrast to the Han groups(P < 0.05-0.001).Gender,age distribution,height,glucose,the proportion of smokers and waist circumference were similarly distributed between both ethnic groups.Further subgroup analyses found that weight,systolic blood pressure,TC,waist circumference,Apo B,the proportion of smokers,BMI,alcohol consumption,TG,diastolic blood pressure,pulse pressure and LDL-C were raised in patients with HLP in contrast to healthy participants in both ethnic groups.Conversely,patients with HLP were found to have lower levels of Apo A1,Apo A1/Apo B ratios and HDL-C in contrast to healthy individuals;these findings were similar irrespective of ethnic group.Interestingly,there was no any stark differences in terms of height,gender,age distribution and glucose in those who had HLP compared to those who did not in both Han and Maonan ethnic groups.2.The allelic and genotypic frequencies of several SNPs were significantly varied between the two ethnic groups(SYTL3 rs6455600,rs2129209 and SLC22A3 rs446809);between the normal and HLP populations in Han(SYTL3 rs6455600,rs2129209,SLC22A3 rs446809 and rs539398)and Maonan groups(SYTL3 rs6455600,SLC22A3 rs446809 and rs539398)(P < 0.05-0.001);respectively.The dominant model of rs446809 SNP appeared was linked to higher HLP-related morbidity risk in the both groups,while the dominant model of rs539298 SNP exerted features of reduced HLP-associated morbidity in both Han and Maonan groups.The dominant model of SYTL3 rs6455600 and SYTL3 rs2129209 SNPs showed a decreased morbidity of HLP only in those of Han ethnicity while the dominant model of SYTL3 rs6455600 SNP only resulted in raised HLP-related morbidity in individuals of the Maonan ethnicity.At the same time,several SNPs were associated with TC(SYTL3 rs6455600,SYTL3 rs2129209 and SLC22A3 rs539298),TG(SLC22A3 rs446809)and LDL-C(SLC22A3 rs446809)levels in the Han while TC(SYTL3 rs6455600,SLC22A3 rs539298),TG(SLC22A3 rs446809)and LDL-C(SLC22A3 rs446809)were significantly related in those of Maonan ethnicity(P < 0.008-0.001 for each,P < 0.008 was determined to be of statistical significance post-Bonferroni correction).3.Several haplotypes were correlated with TC(SYLT3 A-C-A-A,SLC22A3 A-A,C-A,SYTL3-SLC22A3 A-C-A-A-A-A and A-C-A-A-A-G),LDL-C(SLC22A3 A-A,C-A,C-G and SYTL3-SLC22A3 A-C-A-A-A-A),TG(SLC22A3 C-G,SYTL3-SLC22A3 A-C-A-A-C-G and G-T-C-A-C-A)in Han and TC(SYLT3 A-C-A-A,SLC22A3 A-A,A-G,C-G,SYTL3-SLC22A3 A-C-A-A-A-A,A-C-AA-C-G and G-T-C-A-A-A)and LDL-C(SLC22A3 A-A,A-G,C-G and SYTL3-SLC22A3 A-C-A-A-C-G),TG(SLC22A3 C-G and SYTL3-SLC22A3 G-T-C-A-AA)in Maonan ethnic groups(P < 0.0045-0.001 for each,P < 0.0045 was determined to be of statistical significance post-Bonferroni correction).The haplotypes of the SYTL3 A-C-A-A,SLC22A3 A-A,A-G and SYTL3-SLC22A3 AC-A-A-A-A were associated to raised HLP-linked morbidity in both Maonan and Han ethnicities,whereas the haplotypes of the SLC22A3 C-G and SYTL3-SLC22A3 A-C-A-A-C-G conferred protection against HLP-induced morbidity in both these groups.In the Han group,the haplotypes of the SLC22A3 C-A,SYTL3-SLC22A3 A-C-A-A-C-A and G-T-C-A-C-A conferred a protective effect,while the haplotype of SYTL3-SLC22A3 A-C-A-A-A-G was correlated with a raised HLP-morbidity.The haplotypes of SYTL3-SLC22A3 G-T-C-A-A-A and G-T-CA-C-A were linked to raised HLP-related morbidity in the Maonan group only(P < 0.05–0.001,respectively).4.Several significant three-loci models comprising of SNP-SNP(SYTL3 rs9364496,SYTL3 rs6455600 and SLC22A3 rs446809),SNP-environment(SLC22A3 rs446809,rs539298 and BMI ≥ 24 kg/m~2),haplotype-haplotype(SYTL3 A-C-A-A,SLC22A3 A-G and C-G)and haplotype-environment(SLC22A3 A-G,C-G and BMI ≥ 24 kg/m~2),gene-gene(SYTL3-SLC22A3 A-C-AA-A-A,A-C-A-A-A-G and G-T-C-A-C-A),and gene-environment(SYTL3-SLC22A3 A-C-A-A-A-G,G-T-C-A-C-A and BMI ≥ 24 kg/m~2)interactions were detected in those of Han ethnicity(P < 0.001,respectively).Additionally,several different significant three-loci models comprising of SNP-SNP(SYTL3 rs6455600,SLC22A3 rs446809 and rs539298),SNP-environment(SLC22A3 rs446809,rs539298 and BMI ≥ 24 kg/m~2),haplotype-haplotype(SYTL3 A-C-AA,SLC22A3 A-A and A-G)and haplotype-environment(SLC22A3 A-A,A-G and BMI ≥ 24 kg/m~2),gene-gene(SYTL3-SLC22A3 A-C-A-A-C-A,A-C-A-A-C-G and A-T-C-G-C-A),and gene-environment(SYTL3-SLC22A3 A-C-A-A-C-A,AC-A-A-C-G and BMI ≥ 24 kg/m~2)interactions were detected in those of Maonan ethnicity(P < 0.0041–0.001,respectively).5.Unconditional logistic regression analysis for the interactions between these factors suggested that individuals bearing the SYTL3 rs6455600 CT/TT and SLC22A3 rs446809 CA/AA genotypes were of an increased risk of HLP compared to those with the SYTL3 rs6455600 CC and SLC22A3 rs446809 CC genotypes(adjusted OR = 1.319,95% CI = 1.060-1.812,P = 0.008)in the Han group.The carriers of the SLC22A3 rs539298 AG/GG genotype and BMI ≥ 24 kg/m~2(adjusted OR = 0.735,95% CI = 0.482-0.924,P = 0.006),SLC22A3 A-G and C-G(adjusted OR = 0.676,95% CI = 0.523-0.874,P < 0.001),SLC22A3 CG and BMI ≥ 24 kg/m~2(adjusted OR = 0.658,95% CI = 0.408-0.861,P = 0.004),SYTL3-SLC22A3 A-C-A-A-A-G and G-T-C-A-C-A(adjusted OR = 0.762,95% CI = 0.612-0.915,P < 0.001),SYTL3-SLC22A3 G-T-C-A-C-A and BMI ≥ 24 kg/m~2(adjusted OR = 0.649,95% CI = 0.433-0.816,P = 0.011)possessed a reduced HLP risk in the Han group.Thus,it can be seen that the interaction between genes and environmental factors could influence the incidence of hyperlipidemia in the Han population,and the role of genes play a dominant role is dominant.On the other hand,carriers of the SLC22A3 rs446809 CA/AA and SLC22A3 rs539298 AG/GG(adjusted OR = 1.242,95% CI = 1.018-1.522,P <0.001),SLC22A3 rs539298 AG/GG and BMI ≥ 24 kg/m~2(adjusted OR = 1.263,95% CI = 1.122-1.657,P = 0.023),SLC22A3 A-A and A-G(adjusted OR = 1.838,95% CI = 1.609-2.567,P < 0.001),SLC22A3 A-G and BMI ≥ 24 kg/m~2(adjusted OR = 2.360,95% CI = 2.140-2.922,P < 0.001),SYTL3-SLC22A3 A-C-A-A-C-G and BMI ≥ 24 kg/m~2(adjusted OR = 1.649,95% CI = 1.359–1.901,P < 0.001)were of raised HLP risk in the Maonan group.Thus,it can be seen that there are different gene-environmental interactions between the Maonan and Han ethnic group,and environmental factor(BMI ≥ 24 kg/m~2)plays a dominant role in the interaction in Maonan ethnic group,which may be related to the different dietary habits between the Maonan and Han ethnic groups.6.Multivariate regression analysis found that different environmental factors were correlated with different serum lipid levels in Han and Maonan populations.Conclusions: 1.There were differences in serum lipid levels between the Han and Maonan populations.The TC,TG,LDL-C and Apo B levels in the Maonan ethnic group were significantly higher than those in the Han ethnic group,while the HDL-C,Apo A1 and Apo A1/B levels were significantly lower than those in the Han ethnic group.2.The genotype and allele frequencies of SYTL3 rs6455600,SYTL3 rs2129209 and SLC22A3 rs446809 SNPs were significantly different between Han and Maonan ethnic groups,which could partly account for the differences of blood lipid parameters between Han and Maunan ethnic groups.3.Haplotypes could explain more changes in serum lipid parameters(especially TC and LDL-C levels)and the risk of HLP than any single SNP alone.4.There are different gene-gene/environment interactions between Han and Maonan ethnic group,and the role of genes play a dominant role in Han ethnic group but environmental factor(BMI ≥ 24 kg/m~2)plays a dominant role in the interaction in Maonan ethnic group,this may be related to the different dietary habits between Maonan and Han ethnic groups,which indicates that the influence of dietary habits on lipid level cannot be ignored.Part II Associaton of the SYTL3 and SLC22A3 Polymorphisms,Gene-Gene and Gene-Environment Interactions with Coronary Artery Disease and Ischemic StrokeBackground: Coronary artery disease(CAD)and Ischemic stroke(IS)are still the main factors that causing death and disability worldwide.Although the means of prevention and treatment have been improved,the incidence rate is still increasing year by year and has caused serious social and economic burden.Atherosclerosis is the common pathophysiological basis of both diseases.Hyperlipidemia(HLP)is one of the most important independent risk factors of atherosclerosis.Both CAD and IS are complex diseases affected by many factors,such as heredity,environment and even gene-environment interactions.GWASes in European populations and SYTL3 and SLC22A3 gene polymorphisms have been reported to be associated with serum lipid levels.In addition,we also found that SYTL3 and SLC22A3 gene polymorphisms are associated with serum lipid levels in Guangxi Han and Maonan populations.However,the relationship between SYTL3 and SLC22A3 gene polymorphisms and the pathogenesis of CAD and IS has been rarely reported.Objective: To investigate the effects of SYTL3 and SLC22A3 SNPs,genegene/environmental interactions on the incidence of CAD and IS in the Han population in Guangxi.Methods: A case-control study was conducted among the local Han population in Guangxi.A total of 2,269 samples were collected,including 755 subjects in the control group,758 subjects in the CAD group,and 756 subjects in the IS group.Six SNPs(SYTL3 rs9364496,SYTL3 rs6455600,SYTL3 rs2129209,SYTL3 rs9456350,SLC22A3 rs446809 and SLC22A3 rs539298)were selected as study loci.Genotyping was performed by next-generation sequencing techniques.The same questionnaire was used to conduct epidemiological investigation on the subjects,and the serum lipid level,blood glucose and other related biochemical indicators were measured.The differences of genotype and allele frequency distribution between control and case groups were analyzed and further exploring the influence of genotype,haplotype,and gene-gene/environment interaction on the pathogenesis of CAD and IS.At the same time,the correlation between genotype and gene-environment interactions and serum lipid level were analyzed in the control group.Results: 1.Genotype and allele frequencies of SYTL3 rs2129209 and SLC22A3 rs539298 SNPs were significantly different in case compared with the control group.2.SLC22A3 rs539298 SNP was correlated with TC level in the control group,and SLC22A3 rs539298 G allele carriers had lower TC level than SLC22A3 rs539298 A allele carriers.At the same time,SLC22A3 rs539298 has an interactive effect with alcohol consumption in patients with CAD or IS,and which will reduce the incidence of CAD and IS.3.When exploring the relationship of haplotype with the risk of CAD and IS,it was found that SYTL3-SLC22A3 A-C-A-A-A-A,G-T-C-G-C-A,and A-T-A-AC-A haplotypes would increase the risk of CAD,while SYTL3-SLC22A3 A-C-AA-C-G haplotype would reduce the risk of CAD.Meanwhile,the haplotype of SYTL3-SLC22A3 A-C-A-A-A-A and A-T-A-A-C-A would increase the risk of IS,but SYTL3-SLC22A3 A-C-A-A-A-G and A-C-A-A-C-G haplotypes would reduce the risk of IS.In addition,the interactions of SYTL3-SLC22A3 A-C-A-A-A-A and A-T-A-A-C-A haplotypes with age or smoking would increase the risk of CAD;the interactions of SYTL3-SLC22A3 G-T-C-G-C-A haplotype with smoking and BMI would increase the risk of CAD;the interaction of SYTL3-SLC22A3 A-C-AA-C-G haplotype with alcohol consumption would reduce the risk of CAD.In addition,the interactions of SYTL3-SLC22A3 A-C-A-A-G haplotype with gender and alcohol consumption would reduce the risk of IS;the interactions of SYTL3-SLC22A3 A-C-A-A-A with smoking and hypertension would increase the risk of IS;the interactions of SYTL3-SLC22A3 A-T-A-A-C-A with smoking and age would increase the risk of IS;the interaction of SYTL3-SLC22A3 A-C-A-A-C-G haplotype and hypertension would increase the risk of IS,while the interaction with alcohol consumption would reduce the risk of IS.4.When exploring the relationship between six SNPs and serum lipid levels in the control group,it was found that the interaction between SYTL3 rs9364496 and smoking and BMI ≥ 24 kg/m2 will affect TG or TC levels;the interaction between SYTL3 rs6455600 and smoking and BMI ≥ 24 kg/m2 will influence TG level;the interaction between SYTL3 rs2129209 and alcohol consumption had an effect on HDL-C level,and BMI < 24 kg/m2 had an effect on TC level;the interaction between SYTL3 rs9456350 and alcohol consumption had an influence on HDL-C level,and BMI had an influence on TG level;the interaction between SLC22A3 rs446809 and alcohol consumption had an effect on HDL-C level;the interaction between SLC22A3 rs539298 and alcohol consumption had an impact on HDL-C and Apo A1 levels,and BMI had an effect on TC level.In addition,the interaction of SYTL3 rs9364496 GA/GG with BMI ≥ 24 kg/m~2 and smoking would increase TC and TG levels,respectively;the interaction between SYTL3 rs6455600 CT/TT and BMI ≥ 24 kg/m~2 and smoking would increase TG level;the interaction between SYTL3 rs2129209 AC/CC and BMI < 24 kg/m~2 would reduce TC level,while the interaction with alcohol consumption would increase HDL-C level;the interaction between SYTL3 rs9456350 AG/GG and BMI ≥ 24 kg/m~2 would increase TG level,and the interaction with alcohol consumption would increase HDL-C level;the interaction between SLC22A3 rs446809 CA/AA and alcohol consumption would increase HDL-C level;the interaction of SLC22A3 rs539298 AG/GG with alcohol consumption would increase HDL-C and Apo A1 levels,while the interaction with BMI < 24 kg/m~2 would reduce TC level.5.Pearson correlation analysis noticed that 6 SNPs and several environmental factors were correlated with serum lipid level in both CAD and IS groups.Conclusions: 1.SLC22A3 rs539298 AG/GG carriers had a lower serum TC level than SLC22A3 rs539298 AA carriers,which may be partly account for a lower risk of CAD and IS in SLC22A3 rs539298 AG/GG carriers.2.Different interactions between SYTL3-SLC22A3 SNPs and BMI,smoking and alcohol consumption have different effects on lipid levels.At the same time,the interaction between SLC22A3 rs539298 and alcohol consumption would reduce the risk of CAD and IS.Furthermore,haplotypes could explain more changes in the risk of CAD/IS than any single SNP alone,the interactions between haplotype and different environmental factors would produce different effects on the onset of CAD and IS,thus,it can be seen that CAD and IS are multifactorial diseases involving both genetic and environmental factors.Part III Screening of Inflammatory Molecular Markers associated with Ischemic cardiovascular and cerebrovascular DiseasesBackground: Ischemic cardiovascular and cerebrovascular diseases maintained a high risk of the incidence,mortality and disability and also consumed expensive medical expenses,as well as accounting for 30% of the death worldwide.Coronary artery disease(CAD)and ischemic stroke(IS)appears to be strongly dependent on a person’s genotype,hereditary elements are thought to account for approximately 30-60% of CAD and IS cases.As we all know,atherosclerosis is generally regarded as the pathological foundation of CAD and IS.Therefore,the identification of common pathogenic genes of CAD and IS,especially those related to inflammation,which will produce several positive effects for the early prevention and treatment of coronary heart disease and ischemic stroke.As a novel and practical approach for identifying CAD and IS susceptibility genes,a microarray analysis may be helpful for the early diagnosis of CAD and IS.However,the sensitivity and reproducibility of microarray results may be limited.Thus,a comprehensive analysis may be useful to improve the reliability and integrity of the conclusions.In this way,we can more accurately identify the key genes associated with CAD/IS and further exploring the potential biological functions of the key genes.Previous studies have shown that SLC22A3(also known as EMT;EMTH;OCT3,OMIM: 604842,gene ID: 6581,HGNC:10967)is a gene found on chromosome 6q25.3(Exon count: 15)and functions to produce the organic cation transporter 3 that belongs to a family of peripheral membrane proteins and is critical in biogenic histamine synthesis and deactivation.As a potent pro-inflammatory mediator,histamine enhances LDL-C deposition in vascular endothelial cells by inducing cytokines,adhesion molecules,chemokines and other inflammatory mediators.Coupled with the increased permeability of these cells,histamine produces a conducive environment for atherosclerotic plaque formation.Functional studies have shown that SLC22A3 silencing could significantly inhibit histamine synthesis,synthesis of pro-inflammatory mediators(MCP-1,IL-8 and IL-6),mononuclear cell infiltration and may also impair leukocyte-endothelial interaction.A number of studies have showed that the SLC22A3 rs2048327,rs1810126 and rs3088442 SNPs contributed towards depressed CAD risk through downregulation of SLC22A3 transcription and protein levels.It can be seen that SLC22A3 is not only involved in the pathogenesis of coronary heart disease by affecting blood lipid level,but also may be involved in endothelial dysfunction by mediating the synthesis and degradation of histamine,leading to the occurrence and development of CAD and IS.Objective: The gene expression profile datasets of CAD and IS were downloaded from GEO public database,the common differential genes were obtained after integrated analysis,and molecular markers related to CAD and IS were screened by combining with bioinformatics analysis.Methods: In recent years,big data mining has become a novel technology for the research of several certain diseases.Gene chip technology can provide the expression spectrum information of certain diseases,which is a key and practical technology for the research of certain diseases.With the continuous improvement of chip technology,it can provide a variety of research projects such as DNA sequence detection,gene expression profiling,hybrid sequencing and genome library mapping.Typically,after the chip is sequenced,the original data from the chip is uploaded to a designated database for subsequent researchers to download and analyze.In addition to saving considerable research costs,it can also guarantee the continuity of the follow-up research of this topic.Gene Expression Omnibus Database(GEO)is the largest public database under NCBI,collecting a large number of public data sets,and has become one of the essential databases for data mining.There are many methods for chip data mining,the more commonly used are GEO2 R online tool and R language.Researchers can carry out data mining of corresponding diseases according to their own preferences and data mining methods that they are good at.The sensitivity and reproducibility of microarray results may be limited.Thus,a comprehensive analysis may be useful to improve the reliability and integrity of the conclusions.Identifying the common key genes of multiple diseases with common pathogenesis is of great significance for early prevention and treatment of this type of disease.Two CAD datasets(GSE66360 and GSE97320)and another IS dataset(GSE22255)were obtained from the GPL570 Affymetrix Human Genome U133 Plus 2.0 array.The GSE22255 dataset included 20 normal samples and 20 IS samples.An integrated analysis of 53 normal samples and 52 CAD samples from the two CAD datasets was performed.The original files in CEL format were transformed into an expression value matrix using the Affy package in R with the RMA method to normalize the expression values and the SVA method to remove batch differences.The common differentially expressed genes were obtained by intersecting the CAD related differentially expressed genes and IS related differentially expressed genes.The online tool Database for Annotation,Visualization and Integrated Discovery(DAVID)(version 6.8;david.abcc.ncifcrf.gov)was used to annotate the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway and Gene Ontology(GO)enrichment analyses of the common differentially expressed genes.A PPI interaction network of common DEGs was constructed with the Search Tool for the Retrieval of Interacting Genes database(version 11.0;www.string-db.org).Cytoscape 3.7.1(www.cytoscape.org)was applied to visualize the PPI network.Degrees were used to verify the significance of protein nodes in the PPI network.As one of the core components of the PPI network,the network module may have specific biological functions.The Cytoscape software(version 3.61)Molecular Complex Detection(MCODE)plugin was used to identify the most common and largest module clusters with the following parameters: EASE ≤ 0.05,count ≥ 2 and MCODE score > 6.RT-q PCR was used to validate the four significantly dysregulated m RNAs identified by the microarray results and SLC22A3 in the 603 subjects.ROC curve was constructed to infer the predictive value of validated key genes and SLC22A3 for the risk of CAD and IS.Results: 1.Twenty common differentially expressed genes related to CAD and IS were obtained by integrated analysis.The 20 common differentially expressed genes were mainly concentrated in the following signaling pathways: Toll-like receptor signaling pathway;NF-Kappa B signaling pathway;TNF signaling pathway;Nod-like receptor signaling pathway;Nonalcoholic fatty liver disease;Cytokine receptor interaction;Rheumatoid arthritis;Inflammatory bowel disease.2.The PPI network was constructed.After modularization of MCODE and Cytohubba analysis,the top five key genes of degree were identified as follows: CXCL8,degree = 9;JUN,degree = 9;TNF,degree = 9;SOCS3,degree = 8;TNFAIP3,degree = 8.3.The RT-q PCR results revealed that the expression levels of CXCL8 were increased in IS patients than in normal participants and the expression levels of SOCS3,TNF and TNFAIP3 genes were higher in CAD/IS patients than in normal participants.Meanwhile,there was no difference in the expression of JUN between CAD/IS patients and the control group.4.Logistic regression analysis showed that SOCS3,TNF,TNFAIP3,hyperlipidemia,diabetes and smoking increased the risk of CAD,while drinking reduced the risk of CAD.In addition,SOCS3,TNF,TNFAIP3,CXCL8,hypertension,hyperlipidemia,diabetes,smoking and other factors would increase the risk of IS.5.The AUC values of TNF,TNFAIP3,CXCL8,SOCS3,SLC22A3 and SOCS3-SLC22A3 combined diagnostic model were 0.801(95%CI = 0.788-0.859;P < 0.05)with a sensitivity of 82.6% and a specificity of 68.0%;0.755(95%CI = 0.707-0.802;P < 0.05)with a sensitivity of 72.2% and a specificity of 72.7%;0.663(95%CI = 0.622-0.725;P = 0.08)with a sensitivity of 80.0% and a specificity of 60.6%;0.846(95% CI = 0.805-0.887;P < 0.001)with a sensitivity of 87.8% and a specificity of 83.3%;0.861(95%CI = 0.822-0.900;P < 0.001)with a sensitivity of 88.0% and a specificity of 81.7%;0.923(95%CI = 0.884-0.941;P < 0.01)with a sensitivity of 91.9% and a specificity of 92.5% for prediction of CAD risk,respectively.In addition,the AUC values of TNF,TNFAIP3,CXCL8,SOCS3,SLC22A3 and SOCS3-SLC22A3 combined diagnostic model were 0.863(95% CI = 0.828-0.898;P < 0.01)with a sensitivity of 85.7% and a specificity of 77.3%;0.691(95% CI = 0.643-0.740;P < 0.05)with a sensitivity of 66.5% and a specificity of 70.4%;0.832(95% CI = 0.793-0.872;P < 0.001)with a sensitivity of 81.3% and a specificity of 78.0%;0.854(95% CI = 0.814-0.893;P < 0.001)with a sensitivity of 90.4% and a specificity of 81.3%;0.879(95% CI = 0.866-0.931;P < 0.01)with a sensitivity of 88.5% and a specificity of 86.2%;0.931(95% CI = 0.905-0.957;P < 0.01)with a sensitivity of 94.9% and a specificity of 93.1% for prediction of IS risk,respectively.Conclusions: 1.Inflammation-related genes such as TNF,TNFAIP3,CXCL8 and SOCS3 may be molecular markers for early diagnosis of CAD and IS.2.The diagnostic efficiency of SOCS3 and SLC22A3 combined diagnosis model is better than that of SOCS3 or SLC22A3 alone,so the combined diagnosis model can more effectively diagnose early CAD and IS. |
PDF Full Text Request