| Part 1:Screening of differentially expressed genes in ankylosing spondylitis based on RNA-SeqBackgroundAnkylosing spondylitis is a chronic and autoimmuae disease,characterized by recurrent inflammation and progressive ossification,mainly affects axial bones and attachment sites of soft tissues,and is s highly hereditary and induced by multiple factors.The etiology and pathogenesis of the disease are still unclear.Genes including human leukocyte antigen B27(HLA-B27),tumor necrosis factor alpha(TNF-alpha),programmed death factor 1(PD1)and major histocompatibility complex chain related gene A(MICA),have been found to be closely related to the pathogenesis of the disease.These pathogenic genes are hereditary factors in the pathogenesis of ankylosing spondylitis.With the rapid development of gene sequencing technology,RNA sequencing(RNA-Seq)has become a comprehensive,accurate,economic and feasible method for transcriptional analysis.RNA sequencing is the second generation sequencing technology,with higher accuracy,lower cost and higher speed.It is suitable for the known sequence,and needs to be combined with the first generation technology for the unknown sequence.RNA-seq can identify differentially expressed genes(DEGs)and accurately quantify exons and subtypes.There are previous studies used RNA-Seq to identify related genes,but no RNA-Seq on ankylosing spondylitis had been reported.We used RNA sequencing to carry out omprehensive transcriptional analysis between patients with ankylosing spondylitis and healthy people,to identify differentially expressed genes,and to construct functional annotations,protein interaction networks,transcriptional regulatory networks.ObjectiveTo screen differentially expressed genes of ankylosing spondylitis,and construct protein interaction networks and transcriptional regulatory networks,find candidate genes and explore their biological functions and mechanisms.Methods1 LExtraction of total RNA in peripheral blood:homogenate management,extraction of chloroform and purification of RNA.2.RNA quality testing.3.RNA-Seq library preparation.4.Transcriptome sequencing.5.Identification of differentially expressed genes.6.Functional annotations:GO analysis,KEGG signaling pathway analysis.7.Construction of PPI network.8.Construction of special transcriptional regulatory network.Result1.A total of 503 DEGs,including 338 upregulated and 165 downregulated DEGs.2.According to the GO enrichment analysis,the DEGs were significantly enriched in the following GO terms:response to other organisms,inflammatory response,platelet a granule,cellactivation,and cytokine production.KEGG pathway enrichment analysis demonstrated that the DEGs identified in patients with AS were the most significantly enriched in the following signaling pathways:hematopoietic cell lineage,phagosome,measles,cytokine-cytokine receptor interaction.Fc?R?B,FHL2,STAT2 and SOCS3 are associated with osteoclast differentiation signaling pathway,while IFIT1,IFIT3 and RSAD2 are associated with interferon signaling pathway.3.PPI network was consisting of 92 nodes and 76 edges.PPI network revealed that ISG15,HSPB1,MAPILC3A,IFIT1,IFIT3 and SOCS3 were hub proteins.4.A total of 49 TFs which regulated the expression of the top 10 upregulated and downregulated DEGs in AS were identified and the AS-specific transcriptional regulatory network of the top 10 most upregulated and downregulated DEGs was constructed,which consisted of 69 nodes and 127 edges.In this network,OCT1,Pax4,HNF4,NKX2-5,RUNX1 and HNF were the top 6 regulatory factors.Conclusion1.Osteoclast differentiation signaling pathway,interferon signaling pathway and related genes may be associated with pathogenesis of ankylosing spondylitis.2.FcyR?B,FHL2,STAT2 and SOCS3 are differentially expressed genes related to osteoclast differentiation signaling pathway,IFIT1,IFIT3 and RSAD2 are differentially expressed genes related to interferon signaling pathway.Among them,SOCS3,IFIT1 and IFIT3 are hub proteins.3.RUNX1 and OCTl are important transcription factors in ankylosing spondylitis.,they are related to osteoclast differentiation signaling pathway and interferon signaling pathway respectively,and their corresponding genes are also differentially expressed genes.Part 2:Verifcation of differentially expressed genes and reliability analysis of RUNX1 as pathogenicity-related gene BackgroundGene expression is divided into two stages,transcription and translation.Detection of gene expression is detection of specific mRNA and specific proteins,and can be divided into detection of specific mRNA at transcriptional level and detection of specific protein at translation level.Detection of mRNA at transcriptional level includes Northern blot,ribonuclease protection analysis(RPA)and reverse transcription polymerase chain reaction(RT-PCR),each of which has its own advantages and disadvantages.The main method of protein detection is immunological detection,biochemical reaction detection and biological activity detection.The detection of protein is direct validation,and detection of mRNA is indirect validation,there may be regulatory factors in translation stage,which means gene transcription is not necessarily translated.Because time points of detection are different,mRNA was degraded when proteins reach the peak or proteins continuously increase when mRNA reaches the peak,the level of mRNA is not always consistent with the level of proteins.The technology of RT-PCR was initiated by Higuchi et al in 1992,by which target genes can be quantitatively analyzed.Early PCR relies on adding excessive fluorescent dyes and emitting fluorescent signals.The initial template of the target genes can be reflected by the number of fluorescent signals corresponding to cycles.These steps include RNA extraction,reverse transcription,PCR amplification and quantitative analysis.The target genes for validation are differentially expressed genes screened by RNA-Seq sequencing.We always selected genes with relatively high expression and more differences between samples for validation.Amplification efficiency,PCR reagent,GC preference and change of parameters will result in some differences between PCR and RNA-Seq sequencing.It is necessary to exclude the situations of inversion of contrast,low expression of target gene and inconsistency between samples.ObjectiveSample size was expanded,expression of differentially expressed genes in patients with ankylosing spondylitis was tested,and reliability evaluation which includes area under ROC curve,sensitivity and specificity was made to find out candidate genes with high sensitivity and specificity.Methods1.Primer design:primers of Fc?R?B,FHL2,STAT2,SOCS3.IFIT1,IFIT3,RSAD2,RUNX1,OCT1 and internal reference GAPDH were designed.2.Extraction of total RNA in peripheral blood.3.Reverse transcriptase synthesis of cDNA.4.Amplification by SYBR GreenPCR instrument.5.Agarose gel electrophoresis,detection of gene expression and ROC curve analysis.Result1.Between AS group and normal control group,the differences of FcyRIIB,RSAD2 and OCT1 were not significant,the difference of FHL2 were significant but the direction of regulation was contradictory,the difference of STAT2 was not significant and the direction of regulation was contradictory,so they were not reliable differentially expressed genes.The differences of RUNX1,IFIT1,IFIT3 and SOCS3 were significant(P<0.05).RUNX1 was negatively regulated gene,while IFIT1 and IFIT3 and SOCS3 were positively regulated genes.2.mRNA of 4 genes were used to plot ROC curves,area under ROC curve of RUNX1 was 0.935,with sensitivity of 85%and specificity of 90%.Area under ROC curve of IFIT1 was 0.654,with sensitivity of 75%and specificity of 60%.Area under ROC curve of IFIT3 was 0.736,with sensitivity of 60%and specificity of 75%.Area under ROC curve of SOCS3 was 0.703,with sensitivity of 55%and specificity of 85%.The area of RUNX1 was largest,with higher reliability.Conclusion1.There were significant differences of RUNX1,IFIT1,IFIT3 and SOCS3 between AS group and normal control group.2.RUNX1 had the highest reliability,and might be pathogenicity-related gene.Part 3:Correlation analysis between single nucleotide polymorphisms of RUNX1 and ankylosing spondylitisBackgroundThe key genes of ankylosing spondylitis include B27,TNF-a and IL-1.Subtypes of B27 include B2705,B2704 and so on,B2705 is more common among European and North American white people,B2704 is more common among Asian yellow people,loci of-308,-238 and-850 are confirmed to be associated with AS.30735C and 31017G are two pathogenic SNPs of IL-1 located on exon 6.ERAP1,IL-23,OPG,RANKL,FcRL3,FCGR2A and so on are weaker genes of ankylosing spondylitis,the association between these genes and ankylosing spondylitis is mostly confirmed by genetic polymorphisms.RUNX1 is located at chromosome 21q22.12,with full length of 260kbp,it contains 2 promoters involved in transcription.RUNX1 protein is encoded by 12 exons.Among the exons there are two defined domains,the runt homology domain(RHD)and the transactivation domain(TAD).RUNX1 contains runt domain across exon 3-5 and is used to combine with nuclear binding factor CBF and other DNA,heterodimerization is formed by RUNX1 and CBF?.RUNX1 gene is called subunit a and CBF is called subunit ?.Autoimmune diseases contain complex genetic mechanisms.Single nucleotide polymorphisms(SNPs)are important methods for studying the pathogenesis of autoimmune diseases.Studies of SNPs indicate that RUNX1 may be potential candidate gene for autoimmune diseases.PDCD1 gene regulates function of T cell and tolerance of autoantigen,RUNX1-PDCD1 axis is candidate gene for autoimmune diseases,polymorphisms of PDCD1 gene are associated with systemic lupus erythematosus(SLE).The expression of SLC22A4 is specific in blood and immune tissues,and polymorphisms of RUNX1 affect transcriptional efficiency of SLC22A4,SNPs in binding sites of SLC22A4 and RUNX1 increase RUNX1 expression and inhibit SLC22A4 expression,RUNX1-SLC22A4 is associated with rheumatoid arthritis.SNPs in SLC9A3R1 can destroy binding sites of RUNX1,decrease RUNX1 expression,and reduce SLC9A3R1 transcriptional efficiency.Because SLC9A3R1-RUNX1 binding loss may lead to activation of T cells,regulatory effect of RUNX1 on SLC9A3R1 or NAT9 may be predisposing factor for psoriasis.Previous studies mainly focused on interactive genes which contained binding sites with RUNX1,and the association between ankylosing spondylitis and RUNX1 polymorphisms had not been reported,and the mechanism is still unclear.ObjectiveSNPs of RUNX1 were screened by direct sequencing,focusing on detections of exons,promoters and their flanks.The object was to investigate the distribution of SNPs in healthy people and patients with ankylosing spondylitis,to confirm the genotyping of rs2071029 polymorphic loci,to identify alleles and genotype frequencies,and to study the relationship between rs2071029 and ankylosing spondylitis.Methods1.Screening for polymorphisms of RUNX1:primer design by segmentation,extracting DNA from peripheral blood,PCR amplification,whole sequencing of RUNX1 gene,samples were analyzed,and single nucleotide polymorphisms were screened out.2.Distribution of polymorphisms in patients with ankylosing spondylitis and normal controls:7 genotype and allele frequency were selected.Screen out SNPs with statistically significant differences.3.The genotype and allele frequency of rs2071029:extracting peripheral blood,extracting genomic DNA,designing primers for rs2071029,PCR amplification,restriction enzyme digestion,agarose electrophoresis and sequencing analysis,analysis of genotypes and allele frequencies.Result1.According to DNA sequencing,11 single nucleotide polymorphisms were found,2 in regulatory regions,and 9 in non coding regions.7 polymorphisms could be found from NCBI database.Locus-847 was located on 5'-regulatory region.Loci+ 69741,+ 205351,+ 233586,+ 191995,+ 57470 and + 64280 were located on non-coding region.Loci +57470,+64280 and +69741 were located on flanks of exon 5,which belong to runt domain,and had biological significance.Loci +191995,+205351 and +233586 were near C end with low biological significance.2.Distributions of AA,AG and GG genotypes in patients with ankylosing spondylitis were 9.28%,34.02%and 53.61%respectively.Alleles frequencies of A and G were 27%and 73%respectively.Distribution of AA,AG and GG genotypes in normal controls were 5%,21.25%and 73.75%respectively,alleles frequencies of A and G were 16%and 84%respectively.Chi-square test showed that there was no significant difference between observed values and expected values of genotypes from two groups(Chi-square value were 1.58 and 2.22,0.1<p<0.2),which was accorded with H-W law.Compared with normal control group,genotype chi-square value of AS group was 6.400,P<0.05,the difference was statistically significant.Chi-square value of allele frequency was 6.700,P<0.05,the difference was statistically significant.Conclusion1.Small sample was used to screen out single nucleotide polymorphisms in patients with ankylosing spondylitis and normal controls.Ankylosing spondylitis in Han population from Jining district is related to rs2071029 locus,and rs2071029 locus is located on the promoter of RUNX1.2.Distribution of genotype and allele frequencies of rs2071029 locus further confirmed that RUNX1 is a pathogenic-related gene associated with ankylosing spondylitis.3.RUNX1 is a weak gene,and polymorphism of RUNX1 is associated with pathogenesis of ankylosing spondylitis,RUNX1 can be used as target for diagnosis and treatment of ankylosing spondylitis.Part 4:Possible mechanisms of RUNX1 involved in osteoclast differentiation signaling pathwayBackgroundThe incidence of osteopenia or osteoporosis in ankylosing spondylitis is as high as 50-92%,which could lead to kyphosis,low back pain and affect quality of life seriouly.Its mechanism has not been fully elucidated,which may be related to inflammatory response,RANKL signaling pathway and apoptosis-related factors.CDllb can be used as a marker of osteoclasts,tartrate-resistant phosphatase(TRAP),cathepsin K(CAT)and matrix metalloproteinase 9(MMP-9)are also significant genotype markers in the process of osteoclasts differentiation.Many genes and transcription factors are involved in regulation of osteoclast differentiation.Some genes are associated with macrophages and osteoclast precursors,including M-CSF and PU.1.Some genes are related to bone metabolism,including RANKL,RANK,OPG,DC-STAMP,NIK and c-Fos.Some genes are associated with osteoclast activation,including CLCN7,TCIRG1,LTBP3 and so on.Osteoclast differentiation signaling pathway include two types,classical and non classical.M-CSF and RANKL are classical signaling pathways.Without participation of RANKL and M-CSF,other factors can replace their functions and form non-classical signaling pathway,which include vascular endothelial factors,placental and hepatocyte growth factors and inflammatory factors such as IL-1,6,8 and 11.They account for a small proportion of osteoclast differentiation,but play an important role in osteolytic lesions.RANKL and M-CSF are classical pathway.RANKL is secreted by osteocytes and combined with RANK on osteoclast precursors to regulate differentiation and activity of osteoclasts.The combination of RANK and RANKL induces cascade amplification,osteoclast precursor differentiation into osteoclasts,promote bone resorption,osteoblasts secrete OPG,competitive bind with RANK,promote osteoclast apoptosis,prevent excessive bone resorption.The ratio of OPG to RANKL plays an important role in bone mass regulation.RUNX1 is widely expressed in bone marrow hematopoietic stem cells and plays a role of inhibitor in osteoclast differentiation.RUNX1 can be modified by methylation,acetylation and phosphorylation.Methylation is catalyzed by methyltransferase,which can inhibit IL-3,lysozyme,tumor suppressor and so on.After binding with SIN3A,the downstream genes are activated.N-terminal lysine residues mediate acetylation.DNA methylation and histone acetylation are the regulatory factors of bone metabolism.However,there is no report support the idea that epigenetics of RUNX1 is directly involved in osteogenesis and osteoclast.ObjectiveThe expressions of RUNX1,TRAF6 and RANKL with different bone mineral density were measured by reverse transcription polymerase chain reaction(RT-PCR).The correlations of RUNX1.TRAF6 and RANKL were analyzed.The objective was to find the evidence of RUNX1 combined with TRAF6 through immunoprecipitation,and to explore possible mechanisms of RUNX1 involved in signaling pathway of osteoclast differentiation.Methods1.Isolation,culture and identification of osteoclast in bone marrow tissue:cultured in solution and culture plate with 6 hole for 7 days.Light microscope and electron microscope observation.2.Detection mRNA of RUNX1,TRAF6 and RANKL by RT-PCR:designing primers for GAPDH,RUNX1,TRAF6 and RANKL,RNA extraction,synthesis of cDNA,PCR amplification,agarose electrophoresis,gel imaging,and statistically analyzing of relative expression of these genes.3.Detections of RUNX1 combined with TRAF6 by immunofluorescence:Plasmid construction and gene amplification,eukaryotic vector construction,antibody preparation,cell transfection,immunoprecipitation,Western blot test.4.Immunofluorescence:3%paraformaldehyde was immobilized in PBS,free aldehydes,PBS incubation,primary anti-incubation,secondary anti-incubation,Nikon fluorescence microscopy immunofluorescence analysis.Result1.Osteoclasts were observed under light microscope.After staining by tartaric acid,nuclei of the osteoclasts were blue,with 1-3 nuclei and cytoplasm was red.Osteoclasts were observed under electron microscope,it was irregular with diameter of 10-50um,and had pseudo foot and bone resorption lacuna.2.Relative expressions of RUNX1 in three groups of A,B and C were 0.84+0.15,0.47+0.14 and 0.45+0.11 respectively,the difference was statistically significant.Relative expressions of TRAF6 in three groups of A,B and C were 0.74+0.24,1.01+0.32 and 1.04+0.25 respectively,relative expressions of RANKL in three groups of A,B and C were 0.55+0.17,0.47+0.23 and 0.42+0.23 respectively,the differences were not statistically significant.The Pearson coefficient related to RUNX1 and TRAF6 was 0.489,P value was less than 0.05 and the difference was statistically significant.The Pearson coefficient related to RUNX1 and RANKL was 0.372,P value was greater than 0.05 and the difference was not statistically significant.3.FHL2 and TRAF6 were effectively coimmunized with RUNX1,FHL2 and RUNX1 were coimmunized with TRAF6,RUNX1 and FHL2 inhibit the function of TRAF6,which may form complex to participate in formation and differentiation of osteoclasts.4.Immunofluorescence showed that RUNX1 existed mainly in nucleus,TRAF6 existed mainly in cytoplasms,FHL2 existed in both nucleus and cytoplasms,they had close relationship in osteoclasts.Conclusion1.With RUNX1 expression decreasing,osteoclast differentiation is enhanced.Bone destruction of ankylosing spondylitis was aggravated.2.RUNX1 is related to TRAF6 in the process of osteoclast differentiation,but no evidence support that RUNX1 is directly related to RANKL.3.RUNX1,FHL2 and TRAF6 may form immune complex to participate in osteoclast differentiation. |
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