Screening Of Differentially Expressed Circular RNA In The Plasma Of Patients With Tuberculosis And Their Application In Tuberculosis Diagnosis

Background:Tuberculosis?TB?is a chronic infectious and a kind of immune disorder disease,caused by the Mycobacterium tuberculosis?Mtb?infected.It is hard to make effective diagnosis and treatment of TB for lack of biomarker which is highly associated with TB,poses a serious challenge to the effective prevention and control of TB,and is one of the main causses of the global epidemic of tuberculosis.Circular RNA?circRNA?is a newly discovered endogenous RNA,which is evolutionarily conservative and has the specificity of tissue and developmental stage,which is rich and stable in cytoplasm.More and more studies have suggested that circRNA is involved in various human pathological and physiological processes,which can be used as potential diagnostic biomarkers for diseases such as tumors,cardiovascular diseases and neurological diseases.The role of circRNA in the process of anti-mtb infection and whether it can be used as a diagnostic marker for tuberculosis is not yet reported.Objective:In this study,we invested the expression profiles of circRNA in plasma of tuberculosis patients by gene chip technology,to screened and identified the potential of circRNA in tuberculosis diagnosis.Then to explore the correlation between circRNA and the severity of tuberculosis and its ability to evaluate the efficacy of anti-tb treatment.In order to provide a new biomarker for the diagnosis and treatment of tuberculosis.Methed:The expression profiles of circRNA in plasma from 3 patients active pulmonary tuberculosi and 3 healthy controls were analyzed by microarray assay.Six elevated circRNAs?hsa_circ₀009024,hsa_circ₀001953,hsa_circ₀008297,hsa_circ₀003528,hsa_circ₀003524 and hsa_circ₀015879?were selected for clinical validation using real-time fluorescence quantitative PCR?qRT-PCR?in 50 patients with active pulmonary tuberculosis and 50 healthy controls.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.A total of 120 active pulmonary tuberculosis patients,100 health volunteers,40 cases of pneumonia,40 cases of COPD and 40 patients with lung cancer were collected,5ml peripheral blood for each subjects were got and all plasma samples were isolated.Then the expression of hsa_circ₀001953 and hsa_circ₀009024 in each group were detected by qRT-PCR.And the sensitivity and specificity of hsa_circ₀001953 and hsa_circ₀009024 to diagnosis TB were analyzed by ROC curve.The expression levels of hsa_circ₀001953 and hsa_circ₀009024 in the plasma of 25 patients receiving standardized anti-tuberculosis treatment before and after treatment were monitored.Results:1.Sixty-one circRNAs were differentially expressed in plasma of TB patients compare to the healthy control,of which 43 circRNAs were up-regulated and 18 were down-regulated?fold changes ?1.5 and P<0.05?.Results showed that hsa_circ₀009024,hsa_circ₀001953,hsa_circ₀008297,hsa_circ₀003528,hsa_circ₀003524 and hsa_circ₀015879 were obviously increased 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 elevated circRNAs in serving as candidate biomarkers for TB diagnosis,ROC curve analysis was performed for the 6 obviously increased circRNA.The AUC of hsa_circ₀001953,hsa_circRNA₀009024,hsa_circ₀003528,hsa_circ₀003524,hsa_circ₀008297 and hsa_circ₀015879 were 0.856?0.808?0.768?0.715?0.692 and 0.676 respectively.Correlation analysis results showed that hsa_circ₀001953?P<0.001?? hsa_circ₀009024?P=0.001?and hsa_circ₀008297?P=0.027?were correlated with the Severity of Illness Index.While,hsa_circ₀015879?P=0.135??hsa_circ₀003528?P=0.336?and hsa_circ₀003524?P=0.578?were not correlated with the Severity of Illness Index.3.The results shows that the expression levels of hsa_circ₀001953 and hsa_circ₀009024 in plasma of TB patiens were significantly higher than those in healthy controls and disease control groups with expanded clinical samples.The results of ROC curve analysis showed that the AUC of hsa_circ₀001953 to identify TB and healthy persons was 0.826,the sensitivity and specificity was 69.17% and 89.00% respectively.While the AUC of hsa_circ₀009024 was 0.777,The sensitivity and specificity was 60.00% and 86.00% respectively.The AUC of hsa_circ₀001953 combined with hsa_circ₀009024 to diagnosis the patients with TB from healthy controls was 0.915,the sensitivity and the specificity was 72.50% and 96.00% respectively.The AUC of hsa_circ₀001953 combined with hsa_circ₀009024 in the discriminated the patients with TB from dieases controls?pneumonia patients,COPD patients,and lung cancer patients?was 0.897,the sensitivity and the specificity was 79.17% and 86.67% respectively.The levels of hsa_circ₀001953 and hsa_circ₀009024 in the plasma of TB patients was significantly decreased after standard treatment for 6 month.Conclusions:1.Compared to the healthy control,the differentially expression profiles of circRNA in plasma of tuberculosis patients suggested that circRNA may play a important role in the development of tuberculosis.2.Hsa_circ₀001953 and hsa_circ₀009024 were significantly elevated in plasma of TB patients,and were related to the severity of TB.The expression levels of TB patients decreased significantly in plasma after effective treatment.Hsa_circ₀001953 and hsa_circ₀009024 are expected to be a potential biomarkers for tuberculosis diagnosis and curative effect monitoring.