Microarray Expression Profile Of Circular RNAs In Peripheral Blood Mononuclear Cells From Active Tuberculosis Patients

Objective:We aimed to determine circular RNA?circRNA?profile in peripheral blood mononuclear cells?PBMC?from active tuberculosis?TB?patients to identify novel biomarkers for TB.Methods:1.Expression profile of circRNAs in PBMC from 3 active pulmonary TB patients and 3 healthy controls were analyzed by microarray assay.Six circRNAs?the top 3 elevated circRNAs and top 3 decreased circRNAs?were selected for validation using real time-quantitative PCR?qRT-PCR?in 40 TB patients and 40 control subjects.Receiver operating characteristic?ROC?curve was constructed to evaluate their values in TB diagnosis.The correlation between circRNA levels and stages of pulmonary lesions visualized by radiography were calculated using Spearman rank correlation.2.Based on the area under the curve?AUC?of the 6 candidates and their correlations with the disease severity,hsa_circRNA₀01937 was chosen for further evaluation in an independent cohort consisting of 115 TB,40 pneumonia,40 COPD,40 lung cancer patients and 90 control subjects.The levels of hsa_circRNA₀01937 were detected by qRT-PCR.Furthermore,the 115 patients with TB were divided into different subgroups according to cavity formation and the lung fields involved: patients without lung cavity?70 cases?vs those with lung cavity?45 cases?,patients with involvement of <2 lung fields?63 cases?vs ?2 lung fields?53 cases?.An six-month follow up was performed in 20 newly diagnosed TB patients to investigate the expression change of hsa_circRNA₀01937 after chemotherapy.3.To evaluate hsa_circRNA₀01937 potential function,the hsa_circRNA₀01937/miRNA interaction was predicted using Arraystar's home-made mi RNA target prediction software based on TargetScan and miRanda.The expression levels of hsa_circRNA₀01937-target miRNAs were validated by qRT-PCR in 20 patients with TB and 20 healthy controls.The correlation between expression of hsa_circRNA₀01937 and miR-26b-3p was analyzed Pearson rank correlation.Results:1.Thirty-seven circRNAs were differentially expressed between the two groups,of which 13 circRNAs were up-regulated and 24 were down-regulated in TB patients?fold changes ?1.5 and P<0.05?.Results showed that hsa_circRNA₀01937,hsa_circRNA₀09024 and hsa_circRNA₀05086 were increased,while hsa_circRNA₁02101,hsa_circRNA₁04964 and hsa_circRNA₁04296 were decreased in TB patients versus normal control samples?P<0.001?.The results were generally consistent with the microarray data.2.To evaluate the values of these differentially expressed circRNAs in serving as candidate biomarkers for TB diagnosis,ROC curve analysis was performed for each circRNA.The AUC of hsa_circRNA₀01937 reached 0.873,which was the largest among these 6 circRNAs.The AUC of the other circRNAs were 0.810 for hsa_circRNA₀09024,0.765 for hsa_circRNA₀05086,0.782 for hsa_circRNA₁02101,0.824 for hsa_circRNA₁04964 and 0.754 for hsa_circRNA₁04296.Among these circRNAs,hsa_circRNA₀01937,hsa_circRNA₀09024 and hsa_circRNA₁02101 were correlated with the radiological severity scores?RSS?,hsa_circRNA₀05086,hsa_circRNA₁04964 and hsa_circRNA₁04296 were not correlated with the RSS.3.PBMC levels of hsa_circRNA₀01937 in TB patients [2.08?1.45,2.91?] were significantly higher than those in patients with healthy controls [0.98?0.83,1.33?],pneumonia [1.20?0.85,1.50?],COPD [1.05?0.74,1.48?],and lung cancer [0.85?0.69,1.23?]?H=78.42,P<0.0001?.The AUC of hsa_circRNA₀01937 in the discriminated the patients with TB from normal controls,pneumonia patients,COPD patients,and lung cancer patients were 0.849,0.806,0.830,0.879,respectively.PBMC levels of hsa_circRNA₀01937 in cavity pulmonary TB patients were higher than that in pulmonary TB patients without cavity?U=1027,P=0.002?.PBMC levels of hsa_circRNA₀01937 in TB patients with involvement of ?2 lung fields were higher than <2 lung fields?U=1019,P<0.001?.As compared to their pre-treatment [2.47?1.76,3.15?],the hsa_circRNA₀01937 levels decreased significantly in 3 months [1.15?0.92,1.65?]?U=54.00,P<0.001?and 6 months [0.98?0.89,1.40?]?U=30.00,P<0.001?after anti-TB therapy.4.We found that the potential mi RNAs targets of hsa_circRNA₀01937 include miR-22-5p,miR-26b-3p,miR-10b-3p,miR-376a-5p and miR-597-3p.Of the five miRNAs studied,the levels of miR-26b-3p were significantly reduced in TB patients.Correlation analysis showed that the expression of hsa_circRNA₀01937 in PBMC was negatively correlated with miR-26b-3p?r=-0.7185,P=0.004?.Conclusions:1.We identified differentially expressed circRNAs in PBMCs from subjects with active TB and normal controls.These dyregulated circRNAs may play a role in the pathogenesis of tuberculosis.2.Hsa_circRNA₀01937 was significantly elevated in TB patients,and was related to the severity of TB.our data also suggest that hsa_circRNA₀01937 can be used as a potential diagnostic biomarker of TB.3.Hsa_circRNA₀01937 may play a role in the pathogenesis of tuberculosis by functioning as a sponge of the miR-26b-3p.