Research On Exosomal MiRNA As A Biomarker Of Tuberculosis

OBJECTIVE: The aim of this study is to extract the exosome-drived miRNA from THP-1 cell line infected with Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra;then to identify the exosome by western blotting and transmission electron microscopy;high-throughput sequencing and RNA-seq data bioinformation analysis aims to reveal the differentially expressed miRNA;and further select some miRNA to evaluate the clinical value as a potential biomarker of tuberculosis.METHODS:(1)THP-1 cell line were infected with BCG and H37 Ra strains respectively,the uninfected group is Control.After 72 h of culture,exosomes of cell culture supernatants were extracted by ultracentrifugation and identified by western blotting and transmission electron microscopy.(2)After extraction of exosomal total RNA,a new generation of Illumina high-throughput sequencing,RNA-seq data analysis,and multiple bioinformatics methods for differential expression analysis of sequencing were performed.Further,GO analysis,KEGG signal pathway analysis were conducted and target genes were predicted.(3)The serum of tuberculosis patietns and healthy human were used to extract the exosomes,and miRNA was isolated by a kit.Then qPCR was used to verify the expression of differential expressed miRNA in these clinical samples.RESULTS:(1)THP-1 cells showed pseudopodia and macrophage morphology after infected,indicating that the infection was successful;Western blotting showed that the precipitate contained both TSG101 and CD9 exosomal surface proteins,and transmission electron microscopy of the diameter of the precipitate is in the range of 30-150 nm with a cup-shaped bilayer membrane structure.The result validated that the precipatate is exosomes.(2)The concentration of exosomal RNA in the BCG-infected group was 71.74 ng/?L,the concentration of exosomal RNA in the H37Ra-infected group was 68.17 ng/?L,and the concentration of exosomal RNA in the Control group was 62.83 ng/?L;The library was well constructed and detected by the Agilent 2100 Bioanalyzer instrument,which means the sequence can be conducted;The exosomal miRNA of BCGinfected group and the exosomal miRNA of H37Ra-infected group were compared with the exosomal miRNA of control group for differential expression analysis.The |logFC|>1 was used as a rule and after screening,12 miRNA were significant up-regulated,such as hsa-miR-186-5p,hsa-miR-142-3p,hsa-miR-493-3p,hsa-miR-17-5p,hsa-miR-335-3p,hsalet-7e-5p,hsa-miR-185-5p,hsa-miR-146b-5p,hsa-miR-486-5p,hsa-miR-192-5p,hsa-miR-223-3p,hsa-miR-222-3p.Two miRNA were significantly down-regulated,such as hsamiR-548o-3p and hsa-miR-126-5p.According to FC and Reads,miR-142-3p was choosed to be evaluated by the clinical samples.The four target gene prediction software of Targetscan,miRwalk,miRDB and PicTar were used to predict each differential miRNAs target gene and then intersected,there were 1446 target gene for all differential miRNAs;GO analysis and KEGG signal pathway analysis were performed on these target genes.(3)There was a significant difference in the expression of miR-142-3p between tuberculosis patients and healthy people.Conclusion:(1)An exosome isolation and identification technique based on Mycobacterium bovis BCG and Mycobacterium tuberculosis H37 Ra strains infected with THP-1 cell line was established.(2)The successful construction of RNA library,RNA-Seq data analysis showed that BCG infection group and H37 Ra infection group had differentially miRNA expression compared with control group,and some of these miRNAs are the associated with tuberculosis immune signaling pathway.(3)A preliminary clinical evaluation revealed the potential value of miR-142-3p for the diagnosis of active tuberculosis.This study indicate some exosome-drived miRNAs in mycobacterial infection are associated with tuberculosis immune signaling pathway,and miR-142-3p has a potential value as a biomarker of rapid TB diagnosis.The results in this study provide a new perspective for the analysis of the current difficulties in the research of the pathogenesis of tuberculosis and the development of new rapid diagnosis techniques for tuberculosis.