The Potential Diagnostic Value Of Exosome-derived MiR-548o-3p In Active Pulmonary Tuberculosis

Background:Tuberculosis?TB?is a major single infectious disease that causes worldwide death after infection with mycobacterium tuberculosis?MTB?.^[1]The classical TB diagnosis methods has some defects.Exosome is a lipid bilayer extracellular vesicle?EVs?containing different components,including protein,lipid,DNA,mRNA and non-coding RNAs.More and more evidences show that exoskeleton somatic membrane can protect nucleic acid.These vesicles are secreted by almost all cell types and enter the body fluids.In TB,the expression of miRNAs in exosomes in the body fluids of TB patients was up-regulated or down-regulated.The natural or host immune response is then modulated to affect TB progression.Exosome miRNAs can provide a new biomarker for the early and minimally invasive diagnosis of TB because exosomes are present in a variety of body fluids and miRNAs are relatively stable in exosomes.Mir-548 has been reported to be involved in tumor or antiviral immune response in many literatures,but its role in tuberculosis or its diagnostic value as active tuberculosis are still unclear.Objective:A miRNAs with sufficient sensitivity and specificity was expected to be found as a biomarker for the diagnosis of active tuberculosis.Method:1.Isolation of exosomes from Ra/BCG infected thp-1 source macrophages supernatant and identification of exosomes using TEM or WBBefore infection with Ra/BCG strain in logarithmic growth period,THP-1 cells was treated with 200 nM PMA to induce differentiation into adhered macrophages with stronger phagocytosis ability.Then,Ra and BCG were used to stimulate the M?and uninfected cells were used as the control group.After infection for 4 h,the bacteria outside the cells were washed with PBS and added into fresh culture medium to continue culture for 72 h.Cell supernatant was collected from Ra/BCG infection group or control group,and about 35 mL of cell supernatant in each group was used to extract exosomes by ultracentrifugation.After purification of outside secrete weight suspended in PBS save to-80?.The above preserved PBS exosomes were resuspended at 50?L,fixed with 2%paraformaldehyde and negatively stained with1.5%phosphotungstic acid,and their morphology and purity were observed by TEM.At the same time,RIPA lysate was used to resuspend and lyse the excess exosome precipitate,and the identified proteins TSG-101 and CD9 of exosome were detected by Western Blot,respectively.The expression of these proteins in the excess precipitate was analyzed with the skeleton protein?-actin as internal reference.2.Total RNAs extraction,small RNA sequencing and bioinformatics analysis of supernatant exosomes from cultured cells were performedTRIzol reagent was used to lyse and extract total RNA from exosomes,and then the concentration and quality of extracted total RNA were determined.Qualified total RNA samples were sent to Shanghai yunxu company for small RNA sequencing.For small RNAs sequencing,after Illumina sequencing machine sequencing,image analysis,after identification of the bases,harvest after quality control of the original reads,then go to joint,to low quality reads,merge all the sample trimmed reads,new miRNAs projections.And each sample trimmed reads than to merge human pre-miRNAs on the database.The number of tags on each mature miRNA was statistically compared as the original expression level of the miRNA,and the expression level of the miRNA was standardized by TPM.Fold change between the infected group and the uninfected group was calculated,and the differentially expressed miRNAs were screened.Target genes of differentially expressed miRNAs were predicted by miRNA target gene prediction software,and miRNA-target gene network was constructed.GO functional annotation and pathway analysis were conducted on the potential target genes of these differentially expressed miRNAs to find the biological functions or signaling pathways that the differentially expressed miRNAs might play.3.To verify the relative expression levels of miR-548o-3p in plasma exosomes of tuberculosis,lung cancer and healthy groups by RT-qPCRPlasma of 24 patients with tuberculosis,8 patients with lung cancer and 8 healthy volunteers was collected.The exoRNeasy serum/Plasma Midi Kit was used to extract the serum exosomes,followed by the separation of total exosome RNA.Cel-mir-39?1.6×10⁸copies/L?was added to extract the exosome RNA as an external control parameter.Using separated from each secrete body outside,about 60 ng total RNA,according to the miScript?RT Kit instructions for use and its reverse transcription for cDNA tail method,and then the cDNA obtained according to the miScript SYBR Green PCR Kit instructions,join and control cel-miR-39 and objective differences miRNAs specific upstream primer for PCR amplification,it expanded set three parallel hole.Utilization and control cel-miR-39 standardized miRNAs expression,and then use 2^-??Ct calculating miRNAs in TB group outside the plasma secrete body relative to the amount of the expression of tumor or health group.Result:1.Isolation of exosomes from Ra/BCG infected thp-1 source macrophages supernatant and identification of exosomes using TEM or WBTHP-1 cells in stable subculture,using 200 nM concentration of PMA treatment for 24 h,the cell morphology becomes irregular and closely adhered to the wall,indicating that it was successfully induced into differentiation with stronger phagocytosis ability of M?.In Ra/BCG stimulated infection with M?,cell morphology becomes more irregular and aggregation phenomenon is more obvious,acid fast staining shows the presence of mycobacterium in cells,indicating that Ra/BCG infection model construction is successful.TEM detection of exosome precipitation showed that the cup and plate shapes of typical exosomes had a clean background.Western blot assay showed that,compared with the supernatant cells,the precipitated exosome marker proteins CD9 and TSG-101 were highly expressed,while the expression level of internal reference?-actin was not different.2.Total RNAs extraction,small RNA sequencing and bioinformatics analysis of supernatant exosomes from cultured cells were performedThe OD260/280 ratios of total exosome RNA in Ra,BCG infected group and uninfected group were determined by NanoDrop ND-1000 at concentrations of 68.17,71.74 and 62.83 ng/?L,respectively.Sequencing libraries were detected on Agilent2100 Bioanalyzer using Agilent DNA 1000 chip kit.Ra/BCG and uninfected groups were 143,140 and 142 bp libraries with concentrations of 1.26,0.99 and 0.67 ng/?L,respectively.Novel-miRNAs were included in a total of 248?Ra group?,234?BCG group?,and 227?uninfected group?miRNAs.Compared with the uninfected group,12up-regulated miRNAs and 2 down-regulated miRNAs were shown in both Ra and BCG infection groups?reads?10,|log2FC|?1?.Target genes of different miRNAs forecast analysis,and the prediction of target genes GO functional annotations and KEGG pathway analysis,according to the corresponding target genes might miRNAs mainly involved in TGF-?signaling pathways,Ras signaling pathways,PI3K-Akt signaling pathways,Jak-STAT signaling pathways,endocytosis and cGMP-PKG signaling pathways.These signaling pathways in the body's immune and inflammatory response plays a key role,even may affect the progress of the state of tuberculosis.3.To verify the relative expression levels of miR-548o-3p in plasma exosomes of tuberculosis,lung cancer and healthy groups by RT-qPCRThe expression level of plasma exosome miR-548o-3p in the tuberculosis group was up-regulated compared with that in the healthy group,P=0.0404,and was also significantly up-regulated compared with that in the lung cancer control group,P=0.0028.ROC curve analysis showed that AUC=0.869 from tuberculosis group to lung cancer group,and 0.7738 from tuberculosis group to healthy group.Conclusion:1.Typical exosomes with better purity were isolated from both infected and uninfected groups.2.The miRNA library was successfully constructed,and there were differences in miRNA profiles between the Ra/BCG infected group and the uninfected group.The differentially expressed miRNAs in the infected group may regulate the tuberculosis immune response or signaling pathways.3.Plasma exosome hsa-miR-548o-3p has certain value in the differential diagnosis of active tuberculosis.