The Role And Mechamism Of MiR-199a-3p In Transformation And In Inflammatory Response Of Monocyte-macrophage In Atherosclerosis Lesion

Objective:1.To investigate the distribution of content and quantatity of microRNA-199a-3p in atherosclerosis lesion and peripheral blood as well as the possible pathway of its transportation2.To investing the possible pathway of secreting and transporting for the overexpressed miR-199a-3p of macrophage in atherosclerosis lesion,as well as the role in transformation of monocyte in peripheral blood.3.To investigate the impact of miR-199a-3p on the inflammatory response of macrophage in Atherosclerosis lesion.Methods:1.Analyse the content of miR-199a-3p in atherosclerosis lesion via fluorescence in situ hybridization(FISH)、in situ hybridization histochemistry(ISHH)、immunohistochemistry(IHC);Separate human peripheral blood mononuclear cells by Flow cytometry separation technology and extract exosome of peripheral blood plasma via ultracentrifugation.Quantitaty of miR-199a-3p in the blood cells and exosomes is evaluated separately by Realtime Polymerase Chain Reaction(RT-PCR),as well as in THP-1 cells which is co-cultivated with plasma isolated from atherosclerosis patients or healthy volunteers.2.Detect the expression of CD163/HLA-DR/CD68 on the surface of macrophage in Atherosclerosis lesion via immunohistochemistry and double-labelling immunofluorescence,as well as the CD163/Hp/Hb.The THP-1 cells is cultured separatedy in exosome isolated from atherosclerosis or healthy human for 7 days,then detect the expression CD163/Hp/Hb via double-labelling immunofluorescence,Western Blot.To investigate the impact of miR-199a-3p on monocyte-macrophage transformation,miR-199a-3p mimics is transfected into THP-1 cells and the expression of CD68 and CD163 is quantified after 7 days.3.THP-1 cells are transfected with miR-199a-3p mimics and cultured for 7 days.Chemokine receptors Very late antigen-4(VLA-4)/Lymphocyte function-associated antigen-1(LFA-1)and inflammatory factors interleukin-6(IL-6)/tumor necrosis factors-a(TNF-α)of the cultured cells are detected via Realtime-PCR.Results:1.MiR-199a-3p of macrophage in atherosclerosis lesion is statistically upregulated compared with that in control group.Meanwhile,the overexpressed miR-199a-3p is most likely transported into peripheral blood via exosomes secreted from the macrophage.The miR-199a-3p may influence the monocyte in peripheral blood after the exosomes being taken in.2.Detect the expression of CD163/HLA-DR/CD68 on the surface of macrophage in Atherosclerosis lesion via immunohistochemistry and double-labelling immunofluorescence,as well as the CD163/Hp/Hb.The THP-1 cells is cultured separatedy in exosome isolated from atherosclerosis or healthy human for 7 days,then detect the expression CD163/Hp/Hb via double-labelling immunofluorescence,Western Blot.To investigate the impact of miR-199a-3p on monocyte-macrophage transformation,miR-199a-3p mimics is transfected into THP-1 cells and the expression of CD68 and CD163 is quantified after 7 days.3.The expression of chemokine receptors and inflammatory factors in macrophage are suppressed after it is transfected miR-199a-3p mimics.Conclusion:1.Macrophage of atherosclerosis lesion overexpressed miR-199a-3p and transport it into peripheral blood by secreted exosomes to act on the free monocyte.2.Exosome secreted by macrophage of Atherosclerosis lesion which contains the overexpressed miR-199a-3p could be recognized by the monocyte in peripheral blood via CD 163-Hp-Hb pathway,and then promote the monocyte-macrophage transformation.3.The overexpressed miR-199a-3p could down-regulate the inflammatory response of macrophage in atherosclerosis lesion.