| ObjectiveAnkylosing spondylitis(AS)is a form of spondyloarthritis.In the past decade,great progress has been made in the genetic research of AS,which has provided important clues to the etiology of the disease and also led to major therapeutic innovations.Some of these targeted biotherapeutic agents,such as tumor necrosis factor-αinhibitors and interleukin-17 inhibitors,have entered clinical practice.However,AS is still a disease with unknown etiology and easily misdiagnosed due to the lack of specific clinical manifestations.As a mode of post-transcriptional regulation,alternative splicing(ASp)is a potent producer of protein diversity.In recent years,increasing evidence has confirmed that ASp plays a key role in the pathogenesis of a variety of diseases.Through high-throughput sequencing technology,discovering genetic pathogenesis and reliable biomarkers of clinical significance is a popular method for studying various diseases.The aim of this study is to identify the Differentially alternative splicing genes(DASG)that have potential roles in AS disease activity and new bone formation,and to explore the clinical value of splice isoforms of target DASG as potential AS biomarkers.MethodsThe general clinical data,imaging data,biochemical indicators and blood samples of AS patients hospitalized or outpatients in the Department of Rheumatology of the First Affiliated Hospital of Anhui Medical University from January 2021 to January 2022were collected,and age and gender matched healthy people were randomly selected as controls from the Department of physical examination.High-throughput RNA-seq was used to construct the m RNA expression profiles of Peripheral blood mononuclear cells(PBMCs)in 5 AS patients and 3 normal controls.The Differentially alternative splicing events(DASEs)were also analyzed.The target DASGs related to immune and inflammatory pathways of AS were screened by GO and KEGG enrichment analysis and co-regulatory network construction.Quantitative Real-time PCR(q RT-PCR)was used to validate the selected splice isoforms of DASG in clinical samples(29 AS patients with bonebridge,36 AS patients without bonebridge,and 30 normal subjects).The splice sites of DASGs were verified by Sanger sequencing.Finally,SPSS 26.0software was used to analyze the correlation between splice subtypes of DASG and clinical indicators.Results1.RNA sequencing results:A total of 1103 DEGs were obtained,of which 676 were up-regulated and 427 were down-regulated.And 2854 DASEs,of which 1799 were up-regulated and 1055 were down-regulated.2.Comparison of immune cell populations in PBMC between AS patients and normal controls:Deconvolution method was used to identify the types of immune cell population in PBMC of the two groups of subjects,and it was found that plasma cells,monocytes,macrophages M0,resting mast cells,and neutrophils were less in AS patients.However,the proportion of CD8+T cells,eosinophils,follicular helper T cells and innate immune function T cells(γδT cells)increased in AS patients(P<0.05).3.Construction of co-regulatory network:GO analysis showed that a large proportion of DASGs were enriched in DNA-dependent transcriptional pathways.A total of 1707 TFS were intersected with 856 DASGs to obtain 89 degs.By constructing the co-regulatory network of these transcription factors with DEGs and immune cell populations,it was found that resting CD4+T cells,T follicular helper cells,macrophages,monocytes,resting mast cells,neutrophils,plasma cells,eosinophils,CD8+T cells andγδT cells were significantly associated with these transcription factors(P<0.01).4.Screening of target DASG:Through GO and KEGG enrichment analysis,comparison of reads distribution and literature review in Pubmed,we finally selected ZEB2 as the target DASG with good intra-group consistency,large difference between groups and closely related to immunity and inflammation.5.Splicing site verification of target DASG and its splice subtype:Exon skipping(ES)in the splice subtype of ZEB2 was found by generation sequencing.6.Expression of ZEB2Δ4 in AS and normal controls:The expression of ZEB2Δ4 in AS patients was significantly higher than that in normal controls(P=0.014).The expression level of ZEB2Δ4 in the group with bone bridge formation was higher than that in the group without bone bridge formation(P<0.0001),which was significantly higher in patients than in normal controls(P<0.001).7.The expression of ZEB2Δ4 in different disease activity states:ZEB2Δ4expression was statistically different among the four disease activity groups of ASDAScrp(P<0.0001).However,there was no significant difference in ZEB2Δ4expression between BASDAI remission group and BASDAI active group(P=0.114).8.The expression of ZEB2Δ4 in different structural damage states:The expression of ZEB2Δ4 in patients with gradeⅢandⅣsacroiliac joint damage was significantly different(P=0.023).The expression of ZEB2Δ4 in patients with bone bridge formation was significantly higher than that in patients without bone bridge formation(P<0.0001).9.Correlation analysis:The expression of ZEB2Δ4 was correlated with VAS score(P=0.001),ESR(P=0.010),CRP(P=0.001),ASDAScrp score(P<0.0001)and BASDAI score(P=0.001).BASFI(r=0.390,P=0.001),occipital-wall distance(r=0.371,P=0.002),finger-floor distance(r=0.287,P=0.021),bone bridge formation(r=0.469,P<0.0001)and m SASSS score(r=0.540,P<0.0001).10.Regression analysis:The relative expression of ZEB2Δ4 and clinical relevant indicators were used as independent variables,and were put into the linear regression model with ASDAScrp as the dependent variable.Finally,ZEB2Δ4(β=0.073,t=2.052,P=0.045),ESR(β=0.146,t=3.688,P<0.0001),CRP(β=0.340,t=8.177,P<0.0001),VAS(β=0.381,t=7.848,P<0.0001)and BASDAI score(β=0.392,t=8.1778,P<0.0001)were included in the model,R~2value was 0.941,F value was204.263,P value<0.0001.logistic regression analysis showed that ZEB2Δ4(OR=4.716,P=0.008)and X-ray grade of sjoiliac joint(OR=3.251,P=0.007)were independent risk factors for bonebridge formation.The area under the curve(AUC)of ZEB2Δ4 in the diagnosis of bonebridge formation was 0.772,Jordan index was 0.475,the sensitivity was 0.586,and the specificity was 0.889.Conclusion1.The expression level of ZEB2Δ4 in peripheral blood PBMC of AS patients was significantly higher than that of normal people,which was consistent with the sequencing results.2.Bioinformatics analysis showed that ZEB2Δ4 was involved in the regulation of immunity and inflammation.3.ZEB2Δ4 is correlated with a variety of disease activity indicators and structural damage indicators,and is a biomarker for disease activity and prognosis evaluation. |
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