Differential Expression And Bioinformatics Analysis Of Serum LncRNA In Patients With Ankylosing Spondylitis

Background:Ankylosing spondylitis(AS)is a chronic,progressive autoimmune disease mainly involving the axial joints.With the development of the disease,it will eventually become disabled due to spinal fusion and ligament ossification.At present,the pathogenesis and genetic basis of AS are largely unclear,which cannot be completely eradicated,and drugs can only play a temporary role in controlling and relieving the disease.With the rise of high-throughput sequencing technology,a large number of studies have confirmed that lncRNAs can participate in the occurrence and development of a variety of autoimmune diseases,and even some lncRNAs can be used as diagnostic markers for diseases.However,the role of lncRNAs in the pathogenesis of as has rarely been reported,and the expression profile of lncRNAs in the serum of AS patients and whether lncRNAs can be targeted for early diagnosis and treatment of as are unknown.Objective:This study aims to screen the differentially expressed lncRNAs(DElncRNAs)and the differentially expressed mRNAs(DEmRNAs)in the serum of AS patients and normal controls and to explore the function of DElncRNAs and their value in treatment and diagnosis.According to the targeting relationship ofRNAs,the competitive endogenousRNA(ceRNA)network is constructed to explore the regulatory mechanism of the ceRNA network in the progression of AS,which lays a foundation for further research and has important clinical significance for the discovery of potential diagnostic and therapeutic biomarkers of AS.Methods:From May 2021 to November 2021,a total of 23 AS cases(experimental group)and23 healthy controls(control group)were collected.The experimental group was the first onset of AS patients from mangshen.The control group samples were from mangshen.5m L venous blood was collected from the case group and the control group,and the serum was separated.TotalRNA was extracted from the serum samples of 3 patients in the experimental group and 3 patients in the control group,and c DNA libraries of lncRNAs and mRNAs were constructed.High-throughputRNA sequencing was performed to obtain the DElncRNAs and DEmRNAs expression profiles.Furthermore,GO functional enrichment analysis,KEGG,and Reactome signaling pathway enrichment analysis of target genes of DElncRNAs and DEmRNAs were performed by bioinformatics methods.The STRING software was used to analyze the interaction between differential proteins and construct the PPI network.Cytoscape was used to visualize the PPI protein network and perform module analysis and hub gene identification on the PPI network using the MCODE and Cyto Hubba plugins in Cytoscape.The validity of the high-throughput sequencing data was confirmed by quantitative reverse transcription polymerase chain reaction(q RT-PCR)to evaluate the expression of 8 DElncRNAs in serum samples from20 experimental groups and 20 control groups.According to the lncRNA-miRNA-mRNA targeting regulatory relationship,two lnRNAs(MALAT1 and NBR2)that had been verified by q RT-PCR were used to construct a ceRNA network.Spearman correlation was used to analyze the correlation between the expression level of lncRNA and the clinical indicators of AS patients.The receiver operating characteristic(ROC)curve analysis was performed using the p ROC package of R language to evaluate the diagnosticvalue of DElncRNAs in AS.Results:(1)Compared with the control group,a total of 145 DElncRNAs were screened in the experimental group by sequencing analysis,of which 72 were significantly upregulated and 73 were significantly down-regulated.100 DEmRNAs were also identified,of which 49 were significantly up-regulated and 51 were significantly down-regulated.(2)GO function and pathway enrichment analysis showed that DElncRNAs mainly participated in the immune and inflammatory response pathways,such as regulation of protein ubiquitination,major histocompatibility complex class I-mediated antigen processing and presentation,MAPkinase activation,and interleukin-17 signaling pathways.DEmRNAs were mainly enriched in several signaling pathways associated with endoplasmic reticulum stress,including protein processing in the endoplasmic reticulum,unfolded protein response,and ubiquitin-mediated proteolysis.(3)The subsequent q RT-PCR validation results showed that,compared with the healthy control group,the expression levels of MALAT1:24,NBR2:9,lnc-DLK1-35:13,lnc-LARP1-1:1,lnc-AIPL1-1:7,and lnc-SLC12A7-1:16 in the experimental group patients were consistent with the sequencing analysis results.(4)In addition,a ceRNA network was constructed based on the confirmed lncRNAs(MALAT1:24 and NBR2:9)to determine the interactions between lncRNAs,miRNAs,and mRNAs.(5)We further found that MALAT1:24 and NBR2:9 were positively correlated with some clinical indicators.Serum MALAT1:24 expression level was correlated with ESR(r = 0.521,P = 0.019)and BASDAI score(r = 0.711,P < 0.001),serum NBR2:9 expression level was positively correlated with BASDAI score(r = 0.657,P = 0.002)and VAS(r = 0.481,P = 0.032).(6)Finally,ROC curve analysis showed that MALAT1 and NBR2 may be potential biomarkers of AS.Conclusion:Taken together,our study provided a comprehensive overview of lncRNAs and mRNAs expression profiles in the serum of AS patients,and constructed a ceRNA network based on high-throughput sequencing data,providing new insights into the pathogenesis mechanisms of AS.In addition,our study predicts that MALAT1 and NBR2 can be potential targets for the diagnosis and treatment of AS.