Screening Of MicroRNAs Related To Tuberculosis Infection And Prediction Of Target Gene

Tuberculosis (TB), an infectious disease caused by mycobacterium tuberculosis (MTB), is still a leading disease of death in China. MicroRNAs are small, non-coding RNAs that repress protein translation by targeting specific messenger RNAs. Recent work on microRNA expression in virus and bacteria induced diseases has demonstrated their participation in infectious diseases, but the role of microRNA in the TB disease remains unknown.To explore biological behaviors and disease relevance of microRNAs in the development of active tuberculosis (ATB), we investigated expression profile of basal expressed microRNAs as well as MTB PPD-induced microRNAs in ATB patients compared with healthy controls (HC) to sort out specific microRNAs involving in the pathogenesis of active TB. The expression profile of microRNA before and after the challenge of PPD was first measured using microarray in PBMCs isolated from 4 ATB patients and 3 HC subjects. Disease related microRNA expression spectra were generated from the cluster analysis. We also got a series of special microRNAs for further study.We identified 12 miRNAs which basal expression were significantly up-or down-regulated in ATB patients in the microarray results, and further confirmed 5 microRNAs (miR-132*, miR-365, miR-362-3p, miR-572 and miR-630) were up-regulated in 36 ATB patients compared with 36 HC subjects by quantitative real-time PCR (qRT-PCR). The potential targets for those microRNAs were predicted by computational programs MicroCosm. It turns out that these 5 microRNAs have the joint predictive target-OBSCN. We also found that the expression of OBSCN mRNA were down-regulated in ATB patients by qRT-PCR, which indicates that the down-regulation of OBSCN mRNA might be due to the up-regulated of the microRNAs. We successfully cloned miR-365 and miR-630 to the pSuper expression system, and detected their over-expression in transfected HeLa and A549 cells. The expression of OBSCN mRNA was down-regulated in the microRNA over-expressed cells.The remarkably reactive microRNAs during PPD challenge from microarray-based screening were validated in 44 subjects (22 ATB and 22 HC) by qRT-PCR, in which, upon PPD stimulation, miR-155 and miR-155* displayed differential expression between ATB and HC(p<0.05).The receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of the determined PPD-responsive microRNAs. The associated pathway analysis of the intersection of predicted targets of miR-155 and miR-155* indicated that these 2 microRNAs may be included in many critical immune response pathways. MiR-155 and miR-155* exhibited characteristic expression during lymphocytes activation by TB-specific antigen, suggesting that they may play an important role in the cell-mediated immune aberration in the development of tuberculosis disease and can be potential diagnostic markers under the challenge of specific MTB antigens.