| Background:Rapid diagnosis of tuberculosis is an effective measure to prevent the spread of the disease and to treat it timely.But the grim fact is that the gold standard for the diagnosis still depends on the bacteriological experiments,which was developed more than a hundred years ago and with a positive rate of only about 30%.Therefore,there is an urgent need to develop a new sensitive,specific,rapid and safe diagnostic method of tuberculosis.In our previous study of early diagnostic biomarkers of TB we considered the accuracy and safety of the methods,but lacked of in-depth study for the pathological significance of the biomarkers.In the pathogenesis of tuberculosis,although auto-lysosome plays important role in the macrophagic clearance of Mycobacterium tuberculosis,the correlation between the miRNA biomarkers of tuberculosis and the maturation of autophagosome in macrophages have rarely been reported.Therefore,there is a great significance to understand the oceurrence of tuberculosis,through studing the effect of miRNA biomarkers on the formation of auto-lysosomes in macrophages.Methods:We screened the differentially expressed serum miRNAs by Solexa sequencing between tuberculosis patients and healthy controls.We validated these differentially expressed miRNAs in a large number of samples by qRT-PCR.We established a diagnostic model with the validated miRNAs,using forward stepwise multivariate regression.Further,we focused on the process of autophagosome maturation based on the results of bioinformatics analysis.We investigated the molecular biological mechanism of tuberculosis biomarker miR-423-5p,using the experimental methods of qRT-PCR,westem blot,confocal microscopy,electron microscopy,dual luciferase reporter gene system etc..Results:(1)We identified 181 differentially expressed serum miRNAs by Solexa sequencing between tuberculosis patients and healthy controls,of which 93 were up-regulated and 88 were down-regulated.(2)The expression levels of miR-17-5p,miR-20B-5p and miR-423-5p were verified in a large number of samples by qRT-PCR.A diagnostic model of tuberculosis with an area under the curve of 0.908,and a predictive accuracy of 78.18%was established,by combining the three differentially expressed miRNAs.(3)The results of cellular and molecular experiments showed that,the up-regulated miR-423-5p could inhibit the maturation of autophagosomes in macrophages.The inhibition takes place in the stage of autophagosome-lysosome fusion,rather than in the stage of autophagosome degradation.(4)The results of dual luciferase reporter gene experiment showed that Homo VPS33A was the direct target gene of miR-423-5p.The two CUGCCCCUC domains on the 3'-UTR of Homo VPS33A were the interact sites of miR-423-5p.(5)In the PBMCs of pulmonary tuberculosis patients,there was a negative correlation between miR-423-5p and the mRNA level of VPS33A(r=0.37,p=0.02).Conclusions:(1)A rapid,highly accurate,and pathogen non-contacted potential TB diagnostic model was established through combining three tuberculosis specific miRNAs(miRNA-17-5p,miR-20B-5p and miR-423-5p).The area under the curve of the model was 0.908.The predictive accuracy of the model for distinguishing pulmonary tuberculosis patients from healthy controls was 78.18%(the sensitivity was 83.93%,the specificity was 71.44%).The predictive sensitivity of the model was significantly higher than the clinical diagnostic gold standard for tuberculosis(30%)and the new Xpert test recommended by WHO in 2010(70%).(2)The results of the cellular and molecular experiments showed that,in the occurrence of active pulmonary tuberculosis,the up-regulated diagnostic biomarker miR-423-5p played a very important role in inhibiting autophagosome-lysosome fusion in macrophages through down-regulating the expression of VPS33A. |
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