| Objective:To identify the miRNA expression profiles of exosomes from macrophages stimulated by Treponema pallidum(Tp),and elucidate the role and mechnisms of exosomal miR-146a-5p on permeability and monocyte transendothelial migration of vascular endothelial cell.Methods:Tp(Nichols strain)was propagated by intratesticular inoculation of New Zealand male rabbits.Exosomes from Tp stimulated macrophages were isolated with ExoQuick-TCTM reagent,and characterized with transmission electron microscope,western blot and NanoSight analysis.We first performed a comprehensive miRNA profiling in exosomes using microarray analysis.Then RT-qPCR was performed to validate the expression of miR-146a-5p in exosomes.We also isolated plasma exosomes from 20 untreated patients with early syphilis and 20 healthy controls and detected the expression levels of miR-146a-5p in plasma exosomes of two groups by using quantitative RT-qPCR.The transfer of miRNA-146a-5p between macrophage and HUVEC was verified by confocal microscopy and RT-qPCR.We established HUVEC monolayer formation by Transwell,and then measured the monocyte count and the flux of HRP to evaluate the transendothelial migration and the permeability,respectably.Meanwhile,luciferase reporter assay were used to confirm the binding of exosomal miR-146a-5p to the 3'-UTR of JAM-C.The protein level of JAM-C was examined by flow cytometry after the transfection of miR-146a-5p mimic/inhibitor.Moreover,direct knockdown of JAM-C in HUVEC by siRNA,then measured the monocyte count and the flux of HRP to evaluate the transendothelial migration and the permeability.Results:1.The purified exosomes exhibited typical cup-shaped morphologies with a diameter of 40?100 nm on electron microscopy.Western blot showed rich membrance proteins such as CD9,CD63 and CD81 in exosomes.NTA revealed that no significant difference in concentration between the experimental group and the control group.2.A total of 65miRNAs were identified by miRNA array,of which 35 were upregulated and 30 were downregulated.The level of miR-146a-5p was upregulated by 8.07-fold at 12h,7.35-fold at 24h and 17.70-fold at 48h,respectively.Furthermore,we validated that Tp infection increased exosomal miR-146a-5p expression significantly(12h,t=4.44,P<0.01;24h,t=2.79,P<0.05;48h,t=3.10,P<0.05)using RT-qPCR.We also isolated plasma exosomes from 20 untreated patients with early syphilis and 20 healthy controls and confirmed that the expression level of exosomal miR-146a-5p was significantly higher in early syphilis patients(t=2.82,P<0.01).3.After transfection of a fluorescent Cy3-labeled miR-146a-5p mimic,macrophage were co-cultured with HUVEC.The appearance of red fluorescent Cy3 dye in the HUVEC demonstrates that the Cy3-miR-146a-5p mimic was delivered from the macrophage to the recipient HUVEC.After co-culture,the expression levels of miR-146a-5p was significantly increased in the HUVEC(t=2.98,P<0.05).Prior addition(24 h)of the exosomes secretion inhibitor GW4869(10?M)to the macrophage blocked exosomes production and delivery of miR-146a-5p from macrophage into HUVEC,showing no difference in miR-146a-5 expression between the two groups(t=1.11,P>0.05).4.Exosomes from macrophage transfected with miR-146a-5p mimics co-cultured with HUVEC,which could reduce HUVEC monolayer permeability and monocyte transendothelial migration.5.Dual-luciferase reporter assay indicated miR-146a-5p mimic suppressed JAM-C 3 '-UTR luciferase activity.Meanwhile,we confirmed that miR-146a-5p mimic could decrease the expression of JAM-C in HUVEC and miR-146a-5p inhibitor could increase the expression of JAM-C.Moreover,direct knockdown of JAM-C in HUVEC by siRNA significantly decreased the permeability and monocyte transendothelial migration of HUVEC monolayers.Conclusion:1.Purified viable Tp obtained from rabbit animal model.Exosomes derived from macrophage stimulated by Tp can be used in downstream experiments.2.MiR-146a-5p was enriched in exosomes derived from macrophage stimulated by Tp.3.Exosomal miR-146a-5p from macrophage could be transferred to HUVEC.4.Exosomal miR-146a-5p could decrease endothelial permeability and monocyte transendothelial migration by targeting JAM-C. |
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