Screening And Identification Of Molecular Markers For Diagnosis Of Early Spinal Tuberculosis Based On Circular RNA

Objective:Bone tuberculosis is the most common extrapulmonary tuberculosis,and spinal tuberculosis is the most common tuberculosis of bones and joints,accounting for about 50%of bone tuberculosis.Spinal tuberculosis is mainly diagnosed based on clinical manifestations,imaging signs and related laboratory results.However,in the early stage,clinical manifestations,imaging findings and related laboratory tests are short for specificity,which make early diagnosis of spinal tuberculosis quite difficult.CircularRNAs and microRNAs are important ceRNAs discovered in recent years,playing an important role in the regulation of gene expression,with great potential for early diagnosis of molecular markers.There have been related studies and reports on microRNAs in tuberculosis,but there are still no studies on circularRNAs in tuberculosis.In this study,the differentially expressed circularRNAs in peripheral blood of patients with spinal tuberculosis was screened and identified.The efficacy as a molecular marker for early diagnosis of spinal tuberculosis and the possible mechanism in spinal tuberculosis were preliminarily explored.Methods:The content of this research mainly includes the following parts:1.Circular RNA differential expression profiles in peripheral blood of patients with spinal tuberculosis were screened:using the human circular RNA Array 8×15K for spinal tuberculosis case group?n=4?and blank control group?n=3?were tested.And differentially expressed circular RNAs were screened according to fold change?FC?2 folds?and P value?P<0.05?.Due to it met this condition than the large number of differentially expressed genes,circularRNAs with FC?10 or FC?-5 folds and P<0.05 were further screened for statistical description.2.Verification of gene chip by expanded sample size:Because of the small number of gene chips in this experiment,the results might have false positives and instability.Five circular RNAs were randomly selected,used to enlarged-sample-size verifying by real-time PCR in 32 independent samples?case group n=20;control group n=12?.3.Evaluation the early diagnostic efficiency of circRNA 400005 for spinal tuberculosis:Real-time PCR was used to detect the expression of circRNA₄00005 in 32 independent samples?case group n=20;control group n=12?.The test results were converted into two categories?yes or no differential expression?.As well as,the specificity and the sensitivity were detected between spinal tuberculosis patients and healthy controls.At the same time,by means of establishing a receiver operating characteristic?ROC?curve,the area under the curve?AUC?was used to evaluating the diagnostic efficiency of spinal tuberculosis.4.CircularRNA and microRNA expression profiles were combine-analyzed to explore the possible mechanism which circularRNA participates in the pathological process of spinal tuberculosis:Due to circularRNA can function as a microRNA sponge,in this experiment,the same clinical samples?differentially expressed circular RNAs and microRNAs?were combined different assays through the same microRNA response element?MRE?characteristics.Then,the target gene of microRNA was predicted by means of Miranda,MirBase and TargetScan databases,and was used to bioinformatics analysis such as GO,Pathway,and Network.It is explored that circRNA 400005 may play a biological role in spinal tuberculosis by interfering the expression of miR-19a-3p and its target genes.Results:1.Results of differential expression analysis of circular RNA:The quality test results of all samples were all qualified.A total of 1020 differentially expressed circularRNAs were screened?486 with increased expression and 534 with decreased?.70 circularRNAs in further screened?31 with elevated expression and 39 with decreased expression?shown in the paper.2.The results of the expanded sample size verification of the gene chip:CircRNA 400005 expression increased in the case group?P<0.05?,and the results were basically consistent with the chip detection results.3.Assessing the early diagnosis efficiency of circRNA₄00005 for spinal tuberculosis:The sensitivity was 89.4%while the specificity was 61.5%of the early diagnosis for spinal tuberculosis.And the diagnostic efficiency was evaluated by means of establishing the ROC curve with an AUC=78.1%?p<0.05?.4.The results of combined analysis between circularRNA and microRNA expression profiles:?There was a negative correlation between circRNA₄00005 and miR-19a-3p?spearman's rho=0.824,p<0.05?,indicating that circRNA₄00005 regulates the expression of miR-19a-3p via MRE.?Through the prediction of three databases,TNF was found to be one of the target genes of miR-19a-3p.And miR-19a-3p was in a central position in the network analysis.As well as,the target gene function,suggested by GO analysis,mainly involved MAKP kinase activity,cell metabolism regulation and macrophage chemotaxis.At the same time,implied by pathway analysis,the target gene function mainly involved MAKP,TNF and Rap1 signaling pathways.Conclusion1.CircularRNA also has great potential as a molecular marker for early diagnosis of spinal tuberculosis,due to it has better stability than microRNA,verified by the phenomenon that there are a large number of differentially expressed circularRNAs and microRNAs in peripheral blood of patients with spinal tuberculosis.2.The sensitivity and specificity of circularRNA?circRNA₄00005?for spinal tuberculosis patients are moderate.The results indicate that spinal tuberculosis is a complex network involving multi-gene,multi-level gene expression and regulation.And the diagnosis efficiency of single molecular markers is not satisfying.3.CircularRNA?circRNA₄00005?may play a biological role in spinal tuberculosis by regulating the expression of microRNA?miR-19a-3p?and its target genes,which may participate in signal pathways such as MAKP,TNF and Rap1.And it may regulate the expression.of the target gene TNF playing a role in the pathological process of spinal tuberculosis.This conclusion,however,remains to be further verified by experiments.