Based On Plasma Exosome Small RNA Sequencing Screening Combined With Related MiRNAs And Functional Verification

Tuberculosis(Tuberculosis,TB)is a chronic infectious disease worldwide,which has great impact on public health.Exosomes are a novel class of intercellular signaling regulators that contain a large number of micro RNAs(miRNAs)that can be endocytosed by nearby or remote cells and subsequently regulate recipient cells.miRNAs are a category of small molecule RNA that is endogenous,non-coding and highly conserved,whose high specificity and conserved nature improve the accuracy and precision of disease diagnosis.The purpose of this study was to screen differentially expressed miRNAs and predict target genes by small RNA sequencing technology for plasma exosomes of healthy population(HC)and TB population,so as to obtain TB-related miRNAs and susceptibility genes,in order to provide potential targets for TB diagnosis and treatment.The main results are as follows:1.Plasma exosome small RNA-Seq screening for differentially expressed miRNAs.The purpose of this study was to screen the differentially expressed miRNAs in HC and TB populations from the perspective of exosomes.Exosomes in peripheral blood plasma were separated by overspeed centrifugation,and the surface protein markers,particle size and morphology of exosomes were identified by Western Blotting,transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).Application of small RNA-Seq technology for extraction of plasma exosome made contribution to the building of small RNA libraries,TB/HC group for bioinformatics analysis and screening of differentially expressed miRNAs between groups,14 crossover mirnas were obtained by comprehensive analysis.Meanwhile,Target Scan,RNAhybrid and miRanda softwared were used to predict target genes,and the results showed that miRNAs such as miRNA-766-3p and miR-302a-3p could target TB susceptible genes such as NRAMP1,ASAP1 and SP110.DAVID and KOBAS software were further used for GO analysis and KEGG pathway analysis of target genes,which focused on Rap1,m TOR,lysosome and other related signaling pathways.2.Effect of plasma exosome’s differential miRNAs on NRAMP1 in A549 cells.In this study,the phagocytosis of exosomes by A549 cells was survered by laser scanning confocal microscopy,and the expression levels of differentially expressed miRNAs screened in the previous stage were tested by q RT-PCR.The results displayed that the level of differentially expressed miRNAs were up-regulated after exosomes were phagocytosed by A549 cells.In order to verify miRNAs’ transfection efficiency,after transfecting mimics/inhibitors in A549 cells,it was found that miRNAs expression was upregulated after transfection with miR-372-3p/miR-302a-3p/miR-766-3p mimics and downregulated after transfection with inhibitors.Meanwhile,the regulatory relationship between miRNAs and target genes was detected in HEK-293 T cell lines based on the dual luciferase reporter system,and the results displayed that miR-302a-3p and miR-766-3p could inhibit the expression of NRAMP1 by regulating the 3’UTR of NRAMP1 gene.By transfecting miR-766-3p mimics/inhibitor into A549 cell line to verify its regulation of NRAMP1 expression,the results displayed that the expression of NRAMP1 was remarkably decreased after overexpression of miR-766-3p,while the expression of NRAMP1 was basically unaffected after addition of miR-766-3p inhibitor.The cell proliferation and apoptosis of A549 cell line transfected with mimics/inhibitors were detected,and it was found that miR-766-3p inhibitor cell proliferation and promoted cell apoptosis by regulating the expression of NRAMP1.3.Effect of miR-766-3p on survival of intracellular Mycobacterium tuberculosis(Mtb).Clinical samples were used to detect the expression levels of miR-766-3p and NRAMP1 in human peripheral blood,and the results showed that miR-766-3p was correlated with the expression of NRAMP1 and the development of TB.In this study,after infecting A549 cell line with Mtb,it was discovered that the level of miR-766-3p expression was rised,and the expression level of NRAMP1 was down-regulated.Further detection of cell proliferation and apoptosis showed that both miR-766-3p and A549 cell line infected with or uninfected with Mtb inhibited cell proliferation and promoted cell apoptosis by regulating the expression of NRAMP1.These results indicate that miR-766-3p affects the intracellular survival of Mtb by regulating the expression of NRAMP1,which provides a potential target for the diagnosis and treatment of tuberculosis.