| Background:Ankylosing spondylitis(AS)is a chronic progressive autoimmune disease with sacroiliac joints and axial arthropathy of the spine as the main lesions,with heavy socio-economic burden.At present,the differentially expressed genes(DEG)of AS are not fully understood,and the molecular mechanism of its pathogenesis remains to be elucidated.Delays in diagnosis and inadequate treatment methods both advocate a deeper understanding of disease pathogenesis.The purpose of our study is to screen differentially expressed genes through GEO(Gene Expression Omnibus)data set for relevant bioinformatics analysis,and evaluate the expression of key genes in AS through experiments,and then we further explored the relationship between single nucleotide polymorphism(SNP)of key genes ITGB3,CX3CR1,CD247 and AS susceptibility,looking for potential biomarkers,and providing ideas for therapeutic targets and drug development.Method:We selected the GSE25101 and GSE73754 data sets in the GEO database,obtain the expression matrix through the R software"GEOquery"and"Limma"software packages and standardize and correct them,and filter them with|Fold Change|>1.2 and FDR adjust P value<0.05 DEGs.We used DAVID and KEGG online tools for GO function enrichment analysis and KEGG pathway enrichment analysis,and PPI network analysis for retrieval analysis on STRING.In the verification of the differential gene transcription level,we selected from 40 AS patients and 40 healthy controls,matched by age and gender,and the expression of differential genes was detected by a real-time q PCR system.The key gene SNP typing was based on 650 AS patients and 650 healthy control group,the age and gender were matched,and the SNPscan genotyping technology was used for genotyping.Epidata 3.1 was used for data entry,and SPSS 23.0,R 3.6.0 and Haploview 4.2 software were used for statistical analysis.P<0.05 was considered statistically significant.Results:141 DEGs were screened out,of which 85 were up-regulated and 56 were down-regulated:the first 10 up-regulated genes were TP53INP1,PKN2,STAT5B,ZDHHC18,FLJ20273,LOC88523,BAZ2B,CTSA,PPP4R1,C12ORF35;the first 10 down-regulated genes were CD81,IL2RB,HPS6,M6PR,PUF60,HDAC1,CD247,DNMT1,GZMM,ID2.GO function enrichment analysis showed cytolysis,leukocyte migration involved in inflammatory response,neutrophil chemotaxis,blood coagulation,negative regulation of DNA binding.The KEGG pathway mainly focuses on natural killer cell-mediated cytotoxicity,hematopoietic cell lineage,complement and coagulation cascades,transcription disorders in cancer,and the interaction pathways between cytokines and cytokine receptors.In the PPI results,the number of nodes?5 are PRF1,CXCL8,GZMB,IL2RB,KLRD1,CD247,GZMA,PPBP,STAT5B,SPI1,CD7,EOMES,GNLY,KLRB1,NKG7,GZMK,ITGA2B,S100A8,CX3CR1,C5AR1,FCGR3B,GNG11,HDAC1,LPAR2,S100A12,KLRF1,CSF3R,SSTR2,STAT4,CD160,EZR,ID2,ITGB3,TNFSF10.Differential gene q PCR verification of the case-control two groups had no statistical difference in gender(?~2=0.238;P=0.626)and age(t=0.276;P=0.783),and they were comparable.ITGB3 and C5AR1 were up-regulated,CX3CR1,CD247,IL2RB,GZMM and IL8 were down-regulated(P<0.05),but PROS1,PPBP,EIF2AK2 had no difference(P>0.05),the ROC curve analysis showed that ITGB3,CX3CR1 and CD247had high diagnostic value(AUC=0.701,0.709,0.727).The key gene SNP genotyping was successful in 1295 cases,included 646 cases of healthy control and 649 cases of AS,there was no statistical difference in gender and age between the two groups(P>0.05).The SNP analysis results of ITGB3 showed that the distribution of AA,AT,TT genotypes at rs3809865 in the AS group and the control group was statistically significant(?~2=8.913,P=0.012).The results of the dominant model analysis showed that people with AA genotype have a higher risk of AS than those with the other two genotypes(OR=1.400,95%CI=1.106-1.771,?~2=7.867,P=0.005).The overdominant model showed that compared with AT,the risk of AA+TT is also increased(OR=1.450,95%CI=1.134-1.853,?~2=8.846,P=0.003).Besides,the genotype and allele frequency distribution of ITGB3rs4634 were different in different BASDAI groups(?~2=11.959,P=0.003;?~2=6.430,P=0.011).The results of SNP analysis of CD247 showed that the distribution of GG,GC,CC genotypes and allele G and C frequencies of rs3738212 in the AS group and the control group was statistically significant(?~2=7.915,P=0.019;?~2=7.054,P=0.008),the G allele of rs3738212 was associated with an increased risk of AS in the general population and female population(OR=1.320,95%CI=1.075-1.620;OR=1.961,95%CI=1.204-3.193).However,no significant difference was found at each locus of the CX3CR1 gene.Conclusion:The ITGB3,C5AR1,CX3CR1,CD247,IL2RB,GZMM,and IL8 genes may be potential biomarkers of AS;the polymorphisms at the rs3809865 locus of the ITGB3 gene and the rs3738212 locus of the CD247 gene may be related to the genetic susceptibility of AS in the Chinese Han population.The rs4634 locus of ITGB3 gene may be related to BASDAI. |
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