基于高通量芯片对强直性脊柱炎的生物信息学分析
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摘要
目的运用生物信息分析工具筛选出强直性脊柱炎(ankylosing spondylitis,AS)相关的差异表达基因(differentially expressed genes,DEGs)并进行文献挖掘。方法从GEO数据库中检索获取AS患者的芯片数据,通过GEO2R进行DEGs的筛选,运用DAVID数据库对筛选获得的DEGs行基因富集和通路分析,通过STRING数据库构建蛋白相互作用(protein-protein interaction,PPI)网络,并采用Cytoscape软件行进一步分析。结果共筛选出190个差异基因,其中上调的基因75个,下调的基因115个。GO分析显示差异表达基因主要涉及炎症反应、白细胞迁移、胞外区、细胞外结构域、细胞外区域、细胞外基质、脂蛋白颗粒结合和碳水化合物结合等功能簇;KEGG分析提示其在包括类风湿性关节炎和细胞因子-细胞因子受体相互作用等通路中富集;运用STRING数据库构建蛋白相互作用网络;运用Cytoscape筛选出CXCR4、SELL、CD79A、MMP3、CD68、ADIPOQ、CCL19、IL-7R、IL-1β、MYH14、APOE和FCGR3A等12个连接度最高基因,并运用iRegulon插件挖掘得到ETS1、GATA3和IRF8等41个作用于上述12个核心基因的转录因子。结论新发现的12个核心基因和41个转录因子可能在AS的致病机制中起重要作用,并可能成为防治AS的新靶点。
Objective To screen the differentially expressed genes(DEGs) of ankylosing spondylitis(AS) by bioinformatic analysis and make literature review for their functions. Methods The data about genome microarray of AS were retrieved from the Gene Expression Omnibus(GEO) database,and then employed to screen out DEGs between synovial biopsies from AS and undifferentiated spondylitis patients and the samples from healthy individuals and osteoarthritis patients by GEO2 R. The DEGs were then subjected to Gene Ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis in DAVID 6. 8 database. Finally,protein-protein interaction(PPI) network was constructed by STRING10. 5 and then further analyzed by the Cytoscape software. Results A total of 190 differential genes were screened out,of which 75 were up-regulated and 115 were down-regulated. GO analysis showed that the obtained DGEs were mainly involved in inflammatory response,leukocyte migration,extracellular domain,extracellular region, extracellular matrix, lipoprotein particle binding and carbohydrate binding. KEGG analysis suggested that these DGEs were enriched in rheumatoid arthritis and cytokine-cytokine receptor interaction,and others. The PPI network was constructed by STRING database. And then,with the aid of Cytoscape,12 Hub genes with highest degree of ankylosing spondylitis(including CXCR4,SELL,CD79 A,MMP3,CD68,ADIPOQ,CCL19,IL-7R,IL-1β,MYH14,APOE and FCGR3A) were screened out. What's more,41 transcription factors(TFs) interacting with the above 12 Hub genes,including ETS1,GATA3,IRF8 and so on were screened out with the iRegulon,a plugin in Cytoscape. Conclusion The firstly selected12 Hub genes and 41 TFs may play important roles in pathogenesis of AS,and are regarded as new biomarkers for the diagnosis and treatment for AS.
Objective To screen the differentially expressed genes(DEGs) of ankylosing spondylitis(AS) by bioinformatic analysis and make literature review for their functions. Methods The data about genome microarray of AS were retrieved from the Gene Expression Omnibus(GEO) database,and then employed to screen out DEGs between synovial biopsies from AS and undifferentiated spondylitis patients and the samples from healthy individuals and osteoarthritis patients by GEO2 R. The DEGs were then subjected to Gene Ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis in DAVID 6. 8 database. Finally,protein-protein interaction(PPI) network was constructed by STRING10. 5 and then further analyzed by the Cytoscape software. Results A total of 190 differential genes were screened out,of which 75 were up-regulated and 115 were down-regulated. GO analysis showed that the obtained DGEs were mainly involved in inflammatory response,leukocyte migration,extracellular domain,extracellular region, extracellular matrix, lipoprotein particle binding and carbohydrate binding. KEGG analysis suggested that these DGEs were enriched in rheumatoid arthritis and cytokine-cytokine receptor interaction,and others. The PPI network was constructed by STRING database. And then,with the aid of Cytoscape,12 Hub genes with highest degree of ankylosing spondylitis(including CXCR4,SELL,CD79 A,MMP3,CD68,ADIPOQ,CCL19,IL-7R,IL-1β,MYH14,APOE and FCGR3A) were screened out. What's more,41 transcription factors(TFs) interacting with the above 12 Hub genes,including ETS1,GATA3,IRF8 and so on were screened out with the iRegulon,a plugin in Cytoscape. Conclusion The firstly selected12 Hub genes and 41 TFs may play important roles in pathogenesis of AS,and are regarded as new biomarkers for the diagnosis and treatment for AS.
引文
[1] FELDTKELLER E,KHAN M A,VAN DER HEIJDE D,et al.Age at disease onset and diagnosis delay in HLA-B27 negative vs. positive patients with ankylosing spondylitis[J]. Rheumatol Int,2003,23(2):61-66. DOI:10. 1007/s00296-002-0237-4.
[2] STONE M,WARREN R W,BRUCKEL J,et al. Juvenileonset ankylosing spondylitis is associated with worse functional outcomes than adult-onset ankylosing spondylitis[J]. Arthritis Rheum,2005,53(3):445-451. DOI:10. 1002/art.21174.
[3] DEAN L E,JONES G T,MACDONALD A G,et al. Global prevalence of ankylosing spondylitis[J]. Rheumatology(Oxford),2014,53(4):650-657. DOI:10. 1093/rheumatology/ket387.
[4] BAKLAND G,NOSSENT H C,GRAN J T. Incidence and prevalence of ankylosing spondylitis in Northern Norway[J].Arthritis Rheum,2005,53(6):850-855. DOI:10. 1002/art. 21577.
[5] KHAN M A. Epidemiology of HLA-B27 and arthritis[J].Clin Rheumatol,1996,15(Suppl1):10-12. DOI:10.1007/BF03342637.
[6] RADNER H,RAMIRO S,BUCHBINDER R,et al. Pain management for inflammatory arthritis(rheumatoid arthritis,psoriatic arthritis,ankylosing spondylitis and other spondyloarthritis)and gastrointestinal or liver comorbidity[J].Cochrane Database Syst Rev,2012(1):CD008951. DOI:10.1002/14651858. CD008951. pub2.
[7] BREWERTON D A,HART F D, NICHOLLS A, et al.Ankylosing spondylitis and HL-A 27[J]. Lancet,1973,1(7809):904-907.
[8] ZOCHLING J,VAN DER HEIJDE D,BURGOS-VARGAS R,et al. ASAS/EULAR recommendations for the management of ankylosing spondylitis[J]. Ann Rheum Dis,2006,65:442-452. DOI:10. 1136/ard. 2005. 041137.
[9] HEIBERG M S,NORDVAG B Y,MIKKELSEN K,et al.The comparative effectiveness of tumor necrosis factor-blocking agents in patients with rheumatoid arthritis and patients with ankylosing spondylitis:a six-month,longitudinal,observational,multicenter study[J]. Arthritis Rheum,2005,52(8):2506-2512. DOI:10. 1002/art. 21209.
[10] PARK W,YOO D H,MIRANDA P,et al. Efficacy and safety of switching from reference infliximab to CT-P13 compared with maintenance of CT-P13 in ankylosing spondylitis:102-week data from the PLANETAS extension study[J].Ann Rheum Dis,2017,76(2):346-354. DOI:10. 1136/annrheumdis-2015-208783.
[11] FAZEKAS D,KOLTAI M,TREI D,et al. Signa Link 2-a signaling pathway resource with multi-layered regulatory networks[J]. BMC Syst Biol,2013,7:7. DOI:10. 1186/1752-0509-7-7.
[12] JANKY R,VERFAILLIE A,IMRICHOVH,et al. iRegulon:from a gene list to a gene regulatory network using large motif and track collections[J]. PLo S Comput Biol,2014,10(7):e1003731. DOI:10. 1371/journal. pcbi. 1003731.
[13] BAYSAL,DURMU爦B,ERSOY Y,et al. Relationship between psychological status and disease activity and quality of life in ankylosing spondylitis[J]. Rheumatol Int,2011,31(6):795-800. DOI:10. 1007/s00296-010-1381-x.
[14] BROPHY S,CALIN A. Definition of disease flare in ankylosing spondylitis:the patients’perspective[J]. J Rheumatol,2002,29(5):954-958.
[15] DE FILIPPO K,RANKIN S M. CXCR4,the master regulator of neutrophil trafficking in homeostasis and disease[J]. Eur J Clin Invest,2018:e12949. DOI:10. 1111/eci. 12949.
[16] NICOLLE P,LIANG H,REBOUSSIN E,et al. Proinflammatory markers,chemokines,and enkephalin in patients suffering from dry eye disease[J]. Int J Mol Sci,2018,19(4):1221. DOI:10. 3390/ijms19041221.
[17] DASHTI N,MAHMOUDI M,GHARIBDOOST F,et al.Evaluation of ITGB2(CD18)and SELL(CD62L)genes expression and methylation of ITGB2 promoter region in patients with systemic sclerosis[J]. Rheumatol Int,2018,38(3):489-498. DOI:10. 1007/s00296-017-3915-y.
[18] KATIKANENI D S,JIN L. B cell MHC classⅡsignaling:A story of life and death[J]. Human Immunol,2018.[Epub ahead of print]. DOI:10. 1016/j. humimm. 2018.04. 013.
[19] SIEBUHR A S,KJELGAARD-PETERSEN C F,SUN S,et al.Suppression of active,but not total MMP-3,is associated with treatment response in a phaseⅢclinical study of rheumatoid arthritis[J]. Clin Exp Rheumatol,2018,36(1):94-101.
[20]许红飞,初同伟,张超,等. MMP-3在强直性脊柱炎棘上韧带退变中的作用[J].第三军医大学学报,2012,34(19):1957-1960.XU H F,CHU T W,ZHANG C,et al. Role of matrix metalloproteinase 3 in supraspinal ligament degeneration due to ankylosing spondylitis[J]. J Third Mil Med Univ,2012,34(19):1957-1960.
[21] SUZUKI Y,INOUE K,CHIBA J,et al. Histological analysis of synovium by treatment of etanercept for rheumatoid arthritis[J]. Int J Rheumatic Dis,2009,12(1):7-13. DOI:10.1111/j. 1756-185X. 2009. 01372. x.
[22] DIAZ-RIZO V,BONILLA-LARA D,GONZALEZ-LOPEZ L,et al. Serum levels of adiponectin and leptin as biomarkers of proteinuria in lupus nephritis[J]. PLo S ONE,2017,12(9):e0184056. DOI:10. 1371/journal. pone. 0184056.
[23] AKHAVANPOOR M,GLEISSNER C A,GORBATSCH S,et al. CCL19 and CCL21 modulate the inflammatory milieu in atherosclerotic lesions[J]. Drug Des Devel Ther,2014,8:2359-2371. DOI:10. 2147/DDDT. S72394.
[24] QIN Y,HE L D,SHENG Z J,et al. Increased CCL19 and CCL21 levels promote fibroblast ossification in ankylosing spondylitis hip ligament tissue[J]. BMC Musculoskelet Disord,2014,15:316. DOI:10. 1186/1471-2474-15-316.
[25] PONGRATZ G,ANTHOFER J M,MELZER M,et al. IL-7receptorαexpressing B cells act proinflammatory in collageninduced arthritis and are inhibited by sympathetic neurotransmitters[J]. Ann Rheum Dis,2014,73(1):306-312. DOI:10. 1136/annrheumdis-2012-202944.
[26] MONNET D,KADI A,IZAC B,et al. Association between the IL-1 family gene cluster and spondyloarthritis[J]. Ann Rheum Dis,2012,71(6):885-890. DOI:10. 1136/annrheumdis-2011-200439.
[27] WAN Z H,ZHAO Q. Gypenoside inhibits interleukin-1β-induced inflammatory response in human osteoarthritis chondrocytes[J]. J Biochem Mol Toxicol,2017,31(9):e21926. DOI:10. 1002/jbt. 21926.
[28] LEE Y H,BAE S C,SONG G G. FCGR2A,FCGR3A,FCGR3B polymorphisms and susceptibility to rheumatoid arthritis:a meta-analysis[J]. Clin Exp Rheumatol,2015,33(5):647-654.
[29] REDLICH K,KIENER H P,SCHETT G,et al. Overexpression of transcription factor Ets-1 in rheumatoid arthritis synovial membrane:regulation of expression and activation by interleukin-1 and tumor necrosis factor alpha[J]. Arthritis Rheum,2001,44(2):266-274. DOI:10. 1002/1529-0131(200102)44:2 <266::AID-ANR43> 3. 0. CO;2-G.
[30] GOLDRING M B,OTERO M,TSUCHIMOCHI K,et al.Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism[J]. Ann Rheum Dis,2008,67(Suppl3):75-82. DOI:10. 1136/ard. 2008. 098764.
[31] SHAN S,DANG J,LI J,et al. ETS1 variants confer susceptibility to ankylosing spondylitis in Han Chinese[J].Arthritis Res Ther,2014,16(2):R87. DOI:10. 1186/ar4530.
[32]高静.强直性脊柱炎患者外周血Th1/Th2相关转录因子和细胞因子表达水平的研究[D].合肥:安徽医科大学,2012.GAO J. The study on the levels of Th1/Th2 associated transcription factors and some cytokines in patients with Ankylosing spondylitis[D]. Hefei:Anhui Med Univ,2012.
[33] BAI L,LIU Y,HOU S,et al. Association of T-Bet,GATA-3,RORC,and FOXP3 copy number variations with acute anterior uveitis with or without ankylosingspondylitis in Chinese Han[J]. Invest Ophthalmol Vis Sci,2016,57(4):1847-1852. DOI:10. 1167/iovs. 15-17960.
[34] CHENG J,WU R,LONG L,et al. miRNA-451a targets IFN regulatory factor 8 for the progression of systemic lupus erythematosus[J]. Inflammation,2017,40(2):676-687.DOI:10. 1007/s10753-017-0514-8.
[35] LIN J D,WANG Y H,LIU C H,et al. Association of IRF8gene polymorphisms with autoimmune thyroid disease[J].Eur J Clin Invest,2015,45(7):711-719. DOI:10.1111/eci. 12463.
[2] STONE M,WARREN R W,BRUCKEL J,et al. Juvenileonset ankylosing spondylitis is associated with worse functional outcomes than adult-onset ankylosing spondylitis[J]. Arthritis Rheum,2005,53(3):445-451. DOI:10. 1002/art.21174.
[3] DEAN L E,JONES G T,MACDONALD A G,et al. Global prevalence of ankylosing spondylitis[J]. Rheumatology(Oxford),2014,53(4):650-657. DOI:10. 1093/rheumatology/ket387.
[4] BAKLAND G,NOSSENT H C,GRAN J T. Incidence and prevalence of ankylosing spondylitis in Northern Norway[J].Arthritis Rheum,2005,53(6):850-855. DOI:10. 1002/art. 21577.
[5] KHAN M A. Epidemiology of HLA-B27 and arthritis[J].Clin Rheumatol,1996,15(Suppl1):10-12. DOI:10.1007/BF03342637.
[6] RADNER H,RAMIRO S,BUCHBINDER R,et al. Pain management for inflammatory arthritis(rheumatoid arthritis,psoriatic arthritis,ankylosing spondylitis and other spondyloarthritis)and gastrointestinal or liver comorbidity[J].Cochrane Database Syst Rev,2012(1):CD008951. DOI:10.1002/14651858. CD008951. pub2.
[7] BREWERTON D A,HART F D, NICHOLLS A, et al.Ankylosing spondylitis and HL-A 27[J]. Lancet,1973,1(7809):904-907.
[8] ZOCHLING J,VAN DER HEIJDE D,BURGOS-VARGAS R,et al. ASAS/EULAR recommendations for the management of ankylosing spondylitis[J]. Ann Rheum Dis,2006,65:442-452. DOI:10. 1136/ard. 2005. 041137.
[9] HEIBERG M S,NORDVAG B Y,MIKKELSEN K,et al.The comparative effectiveness of tumor necrosis factor-blocking agents in patients with rheumatoid arthritis and patients with ankylosing spondylitis:a six-month,longitudinal,observational,multicenter study[J]. Arthritis Rheum,2005,52(8):2506-2512. DOI:10. 1002/art. 21209.
[10] PARK W,YOO D H,MIRANDA P,et al. Efficacy and safety of switching from reference infliximab to CT-P13 compared with maintenance of CT-P13 in ankylosing spondylitis:102-week data from the PLANETAS extension study[J].Ann Rheum Dis,2017,76(2):346-354. DOI:10. 1136/annrheumdis-2015-208783.
[11] FAZEKAS D,KOLTAI M,TREI D,et al. Signa Link 2-a signaling pathway resource with multi-layered regulatory networks[J]. BMC Syst Biol,2013,7:7. DOI:10. 1186/1752-0509-7-7.
[12] JANKY R,VERFAILLIE A,IMRICHOVH,et al. iRegulon:from a gene list to a gene regulatory network using large motif and track collections[J]. PLo S Comput Biol,2014,10(7):e1003731. DOI:10. 1371/journal. pcbi. 1003731.
[13] BAYSAL,DURMU爦B,ERSOY Y,et al. Relationship between psychological status and disease activity and quality of life in ankylosing spondylitis[J]. Rheumatol Int,2011,31(6):795-800. DOI:10. 1007/s00296-010-1381-x.
[14] BROPHY S,CALIN A. Definition of disease flare in ankylosing spondylitis:the patients’perspective[J]. J Rheumatol,2002,29(5):954-958.
[15] DE FILIPPO K,RANKIN S M. CXCR4,the master regulator of neutrophil trafficking in homeostasis and disease[J]. Eur J Clin Invest,2018:e12949. DOI:10. 1111/eci. 12949.
[16] NICOLLE P,LIANG H,REBOUSSIN E,et al. Proinflammatory markers,chemokines,and enkephalin in patients suffering from dry eye disease[J]. Int J Mol Sci,2018,19(4):1221. DOI:10. 3390/ijms19041221.
[17] DASHTI N,MAHMOUDI M,GHARIBDOOST F,et al.Evaluation of ITGB2(CD18)and SELL(CD62L)genes expression and methylation of ITGB2 promoter region in patients with systemic sclerosis[J]. Rheumatol Int,2018,38(3):489-498. DOI:10. 1007/s00296-017-3915-y.
[18] KATIKANENI D S,JIN L. B cell MHC classⅡsignaling:A story of life and death[J]. Human Immunol,2018.[Epub ahead of print]. DOI:10. 1016/j. humimm. 2018.04. 013.
[19] SIEBUHR A S,KJELGAARD-PETERSEN C F,SUN S,et al.Suppression of active,but not total MMP-3,is associated with treatment response in a phaseⅢclinical study of rheumatoid arthritis[J]. Clin Exp Rheumatol,2018,36(1):94-101.
[20]许红飞,初同伟,张超,等. MMP-3在强直性脊柱炎棘上韧带退变中的作用[J].第三军医大学学报,2012,34(19):1957-1960.XU H F,CHU T W,ZHANG C,et al. Role of matrix metalloproteinase 3 in supraspinal ligament degeneration due to ankylosing spondylitis[J]. J Third Mil Med Univ,2012,34(19):1957-1960.
[21] SUZUKI Y,INOUE K,CHIBA J,et al. Histological analysis of synovium by treatment of etanercept for rheumatoid arthritis[J]. Int J Rheumatic Dis,2009,12(1):7-13. DOI:10.1111/j. 1756-185X. 2009. 01372. x.
[22] DIAZ-RIZO V,BONILLA-LARA D,GONZALEZ-LOPEZ L,et al. Serum levels of adiponectin and leptin as biomarkers of proteinuria in lupus nephritis[J]. PLo S ONE,2017,12(9):e0184056. DOI:10. 1371/journal. pone. 0184056.
[23] AKHAVANPOOR M,GLEISSNER C A,GORBATSCH S,et al. CCL19 and CCL21 modulate the inflammatory milieu in atherosclerotic lesions[J]. Drug Des Devel Ther,2014,8:2359-2371. DOI:10. 2147/DDDT. S72394.
[24] QIN Y,HE L D,SHENG Z J,et al. Increased CCL19 and CCL21 levels promote fibroblast ossification in ankylosing spondylitis hip ligament tissue[J]. BMC Musculoskelet Disord,2014,15:316. DOI:10. 1186/1471-2474-15-316.
[25] PONGRATZ G,ANTHOFER J M,MELZER M,et al. IL-7receptorαexpressing B cells act proinflammatory in collageninduced arthritis and are inhibited by sympathetic neurotransmitters[J]. Ann Rheum Dis,2014,73(1):306-312. DOI:10. 1136/annrheumdis-2012-202944.
[26] MONNET D,KADI A,IZAC B,et al. Association between the IL-1 family gene cluster and spondyloarthritis[J]. Ann Rheum Dis,2012,71(6):885-890. DOI:10. 1136/annrheumdis-2011-200439.
[27] WAN Z H,ZHAO Q. Gypenoside inhibits interleukin-1β-induced inflammatory response in human osteoarthritis chondrocytes[J]. J Biochem Mol Toxicol,2017,31(9):e21926. DOI:10. 1002/jbt. 21926.
[28] LEE Y H,BAE S C,SONG G G. FCGR2A,FCGR3A,FCGR3B polymorphisms and susceptibility to rheumatoid arthritis:a meta-analysis[J]. Clin Exp Rheumatol,2015,33(5):647-654.
[29] REDLICH K,KIENER H P,SCHETT G,et al. Overexpression of transcription factor Ets-1 in rheumatoid arthritis synovial membrane:regulation of expression and activation by interleukin-1 and tumor necrosis factor alpha[J]. Arthritis Rheum,2001,44(2):266-274. DOI:10. 1002/1529-0131(200102)44:2 <266::AID-ANR43> 3. 0. CO;2-G.
[30] GOLDRING M B,OTERO M,TSUCHIMOCHI K,et al.Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism[J]. Ann Rheum Dis,2008,67(Suppl3):75-82. DOI:10. 1136/ard. 2008. 098764.
[31] SHAN S,DANG J,LI J,et al. ETS1 variants confer susceptibility to ankylosing spondylitis in Han Chinese[J].Arthritis Res Ther,2014,16(2):R87. DOI:10. 1186/ar4530.
[32]高静.强直性脊柱炎患者外周血Th1/Th2相关转录因子和细胞因子表达水平的研究[D].合肥:安徽医科大学,2012.GAO J. The study on the levels of Th1/Th2 associated transcription factors and some cytokines in patients with Ankylosing spondylitis[D]. Hefei:Anhui Med Univ,2012.
[33] BAI L,LIU Y,HOU S,et al. Association of T-Bet,GATA-3,RORC,and FOXP3 copy number variations with acute anterior uveitis with or without ankylosingspondylitis in Chinese Han[J]. Invest Ophthalmol Vis Sci,2016,57(4):1847-1852. DOI:10. 1167/iovs. 15-17960.
[34] CHENG J,WU R,LONG L,et al. miRNA-451a targets IFN regulatory factor 8 for the progression of systemic lupus erythematosus[J]. Inflammation,2017,40(2):676-687.DOI:10. 1007/s10753-017-0514-8.
[35] LIN J D,WANG Y H,LIU C H,et al. Association of IRF8gene polymorphisms with autoimmune thyroid disease[J].Eur J Clin Invest,2015,45(7):711-719. DOI:10.1111/eci. 12463.