Expression Profile And Potential Function Of Circular RNA In Peripheral Blood Mononuclear Cells Of Patients With Ankylosing Spondylitis

Background:Ankylosing spondylitis（AS）is one of chronic inflammatory diseases which mainly affects the axial joints.The onset of AS is insidious and the progress is slow.It is common in young and middle-aged men.Patients often suffer from limited physical activity and even disability due to the inability to obtain timely and effective treatment.At present,the pathogenesis of AS is still unclear.Most scholars believe that it is mainly related to genetics,immunity,environment,bacterial infections,etc.,among which genetic factors occupy the dominant position.Circular RNA（circRNA）is a type of closed circular non-coding RNA（ncRNA）.Its main biological function is to combine with microRNA（miRNA）through miRNA response elements（MREs）to relieve miRNA’s inhibitory effect on target genes,thereby regulating the expression of target genes.In recent years,it had been reported that circRNAs can participate in the occurrence and development of tumors,autoimmune diseases,cardiovascular diseases,etc.,and had the potential to become their diagnostic markers.At present,there were few reports related to AS and circRNAs,and whether it is involved in regulating the occurrence and development of AS remains unclear.Therefore,in this study,by analyzing the expression profile of circRNAs in peripheral blood mononuclear cells（PBMC）of patients with AS,and identifying differentially expressed circRNAs,we further elucidate whether circRNAs are involved in the occurrence and development of AS.Objective:To investigate the differential expression of circRNAs in PBMCs of AS patients and healthy control（HC）,to discover AS biomarkers with potential diagnostic efficiency and disease activity judgment,and to explore how circRNAs participate in the regulation of AS pathogenesis.Methods:1.The circRNA microarray technology was used to detect the expression level of circRNAs in 6 ASand 6 HC,and then the circRNA expression profile was established.Fold change（FC）and P value were used to screen out circRNAs with significant differential expression.Gene Ontology（GO）analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis were used to obtained the enriched biological functions and related signal pathways.2.60 AS（30 cases of AS in active stage and 30 cases in stable stage）and 30 HC peripheral blood samples and their clinical data were collected,PBMCs from each sample were isolated,total RNA was extracted,and reverse transcribed into cDNA.Real-time quantitative polymerase chain reaction（RT-qPCR）was performed to detect the relative molecular expression of 5 differentially expressed circRNAs（hsa_circRNA₁04633,hsa_circRNA₀01544,hsa_circRNA₁02532,hsa_circRNA₀08961,hsa_circRNA₀12732）.At the same time,the miRNA target prediction software was used to predict the interaction between the 5 circRNAs and miRNAs and their binding sites.Mann-Whitney U test was used to test the difference of measurement data between two groups,Spearman correlation analysis was used for correlation between variables,and receiver operating characteristiccurve（ROC）curve was used to evaluate biomarkers diagnostic performance of the substance.Results:1.The results of circRNA microarray showed that more than 1300 circRNAs were differentially expressed between AS group and HC group,of which 675 were up-regulated and 693 were down-regulated;GO analysis showed that the differentially expressed circRNAs were mainly located in cells,and it was related to protein kinase activity and cell metabolism.KEGG pathway analysis found that the differentially expressed circRNAs were mainly enriched in related signaling pathways such as inflammation and immunity.2.The relative molecular expression of circRNAs was compared between AS group and HC group.The results showed that compared with HC group,hsa_circRNA₁04633（Z=-4.708,P<0.0001）,hsa_circRNA₀01544（Z=-3.535,P<0.001）and hsa_circRNA₁02532（Z=-2.183,P<0.05）were significantly increased in AS group,while the other circRNAs had no statistical significance between AS group and HC group（P>0.05）.The AS group was further divided into AS active（ASA）group and AS stable（ASS）group according to the inflammatory stage,and then compared with the HC group.The results showed that compared with the HC group,hsa_circRNA₁04633 and hsa_circRNA₀01544 were significantly increased in ASA group（Z=-3.947,P<0.0001;Z=-3.489,P<0.001）and ASS group（Z=-4.184,P<0.0001;Z=-2.617,P<0.05）,and there was no statistical difference between the ASA group and the ASS group（P>0.05）.It was also found that compared with the HC group,hsa_circRNA₁02532 and hsa_circRNA₀08961（Z=-2.292,P<0.05;Z=-2.099,P<0.05）only increased in the ASA group but had no difference between the ASA and ASS groups（P>0.05）.In addition,the difference of hsa_circRNA₀12732 between ASA and ASS groups was statistically significant（Z=-2.188,P<0.05）,and the remaining circRNAs showed no significant difference in each group（P>0.05）.Spearman correlation analysis showed that the relative molecular expression of hsa_circRNA₁04633 was negatively correlated with bath ankylosing spondylitis disease activity index（BASDAI）（r=-0.264,P<0.05）,white blood cell（WBC）（r=-0.271,P<0.05）and granulocyte（GR）（r=-0.267,P<0.05）.The expression level of hsa_circRNA₀12732 was negatively correlated with BASDAI（r=-0.284,P<0.05）,bath ankylosing spondylitis functional index（BASFI）（r=-0.279,P<0.05）,hypersensitive C-reactive protein（hsCRP）（r=-0.334,P<0.01）and globulin（GLOB）（r=-0.431,P=0.001）;while it was positively correlated with lymphocyte（LY）（r=0.260,P<0.05）and albumin（ALB）（r=0.307,P<0.05）.In addition,hsa_circRNA₀08961 was found to be negatively correlated with platelet（PLT）（r=-0.334,P<0.05）.The results of ROC curve analysis found that the area under curve（AUC）（95%CI）of hsa_circRNA₁04633,hsa_circRNA₀01544 and hsa_circRNA₁02532 were 0.806（0.707-0.904）,0.720（0.610-0.831）,and 0.642（0.521-0.762）,respectively.Prediction of target miRNAs for these 5 circRNAs,taking hsa_circRNA₁04633 as an example,its target miRNAs included hsa-miR-22-5p,hsa-miR-24-3p,hsa-miR-423-3p,hsa-miR-452-3p,hsa-miR-653-3p,etc.Conclusion:There were differentially expressed circRNAs in PBMCs of AS patients,and they may be involved in the occurrence and development of AS by regulating inflammation,immune and other related pathways.Among them,hsa_circRNA₁04633 and hsa_circRNA₀01544 have the potential to become the diagnostic molecular markers of AS.