| Research background:Ankylosing spondylitis?AS?is a chronic,progressive inflammatory disease that infringes the axial joints mainly.Inflammation is the most prominent feature of AS,and the most fundamental pathological factor causing new bone formation and spinal rigidity in AS patients.Long noncoding RNA?lncRNA?is a functional RNA molecules that are longer than 200 nucleotides and cannot encode proteins.A large number of studies have shown that lncRNA play important regulatory roles in the occurrence and development of many human diseases,but it is still unknown whether lncRNA may be a molecular marker for the diagnosis of AS and evaluation of disease activity.Objective:To understand the differential expression?DE?of lncRNAs and mRNAs in peripheral blood mononuclear cells?PBMC?of AS patients with different disease activity.In Further,to explore preliminarily the possibility of lncRNAs to be the biomarker for the diagnosis and disease activity of AS.Methods:?1?The PBMCs were collectedfrom 3 active AS patients?AAS,BASDAI>6,or 6>BASDAI>4 and ESR>22mm/h,6>BASDAI>4 and hsCRP>9mg/L?,3 inactive AS patients?IAS,BASDAI<4?and healthy controls?HC?,and microarray was performed to investigate the differently expressed lncRNAs and mRNAs in AS patients,GO analysis and path analysis predicted the roles of differentially expressed mRNAs;Expression of seven lncRNAs were measured using RT-qPCR to verify the data of the microarray.?2?The expression of four lncRNAswhich were screened from seven lncRNAshave been verified according to the difference observed between AS and HC while no difference between AAS and IAS were detected by RT-qPCRamong 30 AAS,30 IAS and 30 HC patients.Spearman's correlation analysis was used to detect the correlation between the four lncRNAs and clinical inflammatory indicators.ROC?receiver operating characteristic curve?analysis was used to analyze their diagnostic value according to the AUC?area under the curve?.?3?The expression of three lncRNAswhich were screened from seven lncRNAshave been verified according to the difference observed among AAS,IAS and HC while no difference between IAS and HCwere detected by RT-qPCRamong 30 AAS,30 IAS and 30 HC patients.?4?The expression level of serum high-mobility group protein B1?HMGB1?was measured using ELISA among 30 AAS,30 IAS and 30 HC.?5?The correlations between the three lncRNAs level and clinical data,HMGB1 level were analyzed by Spearman's correlation analysis.Results:?1?The microarray analysis showed that,2967 and 3137lncRNAs,1597 mRNAs and 2953 mRNAs were differentially expressed in AAS group and IAS group compared with HC group,respectively.Compared with the IAS group,2190 lncRNAs and 1252 mRNAs were differentially expressed in the AAS group,respectively.Bioinformatics analysis showed that the differentially expressed genes were mainly involved in biological processes related to immunity.RT-qPCR verified the reliability of the microarray analysis results.?2?Expression levels of NR037662 and ENST00000599316 in the AAS group and IAS group were higher than those in the HC group[NR037662:1.40?1.42?×10-4vs1.53?1.60?×10-4vs0.72?0.46?×10-4,P<0.001;ENST00000599316:5.43?4.72?×10-3vs8.10?6.45?×10-33 vs3.06?2.26?×10-3,P<0.001],while no difference was observed between AAS and IAS.However,the expression levels of ENST00000577914 and ENST00000579003 were significantly lower than those in the HC group[ENST00000577914:3.06?5.15?×10-5vs2.86?2.04?×10-55 vs4.61?5.77?×10-5,P<0.05;ENST00000579003:1.54?2.42?×10-33 vs 2.36?3.85?×10-33 vs 3.15?3.43?×10-3,P<0.05],while no difference was observed between AAS and IAS.The four lncRNAs were not correlated with the clinical inflammatory indicators?P>0.05,respectively?.The results of ROC curve analysis showed that the AUCs of NR037662,ENST00000599316,ENST00000577914 and ENST00000579003 were 0.804,0.812,0.706 and 0.698,respectively;?3?The expression levels of NR003542and ENST00000512051 in AAS group were higher than those in IAS group and HC group[NR003542:1.33?1.54?×10-22 vs 1.02?0.99?×10-2vs1.03?0.34?×10-2,P<0.05;ENST00000512051:1.45?1.29?×10-22 vs0.96?1.06?×10-22 vs 1.02?0.64?×10-2,P<0.05],while expression level of NR026756 in AAS group was much lower than that in IAS group[0.15?0.58?×10-33 vs 0.50?0.95?×10-33 vs 0.61?0.91?×10-3,P<0.05]and HC group[0.15?0.58?×10-3vs 0.61?0.91?×10-3,P<0.001].The plasma HMGB1expression level in AAS group was higher than that in IAS group and HC group?162.27±172.40ng/ml vs 79.62±40.84ng/ml vs 41.94±25.19ng/ml,P<0.01;respectively?,and was also significantly higher in IAS group than HC group?P<0.01?.The relative expression level of NR003542 were positively correlated with the levels of BASFI,ESR and hsCRP?P<0.05;respectively?,while the relative expression level of NR026756 were negatively correlated with the levels of BASDAI,BASFI,ESR,hsCRP,GLOB and plasma HMGB1?P<0.05;respectively?.There were no significant correlations between ENST00000512051 and all the clinical indicators?P>0.05?.Conclusion:?1?.The differential expression of lncRNA and mRNA in PBMCs of AS patients might be involved in the pathogenesis of AS,and bioinformatics analysis revealed that differentially expressed genes were mainly involved in immune-related biological processes.?2?.LncRNA-NR037662,ENST00000599316 and ENST00000577914might be as the biomarkers for the diagnosis of AS.?3?.LncRNA-NR003542 and NR026756might be as the indicators to evaluate the disease activity of AS. |
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