The Role And Mechanisms Of MiR-127-3p In Regulating Macrophagic Lipid Anabolism And Its Implications In Carotid Atherosclerosis

ObjectiveCarotid atherosclerotic plaques formation and development is one of the leading causes of ischemic cerebral stroke.Previous studies showed that miR-127-3p is significantly upregulated in carotid atherosclerotic plaques.This study aims to investigate the role and mechanisms of miR-127-3p in the formation,development of carotid atherosclerotic plaque,and to provide novel therapeutic target for inhibiting the formation and development of carotid atherosclerotic plaque;thereby providing novel ideas for the research on the mechanisms,through which atherosclerosis(AS)occurs,develops.MethodsThis project covers four aspects: Assay of clinical samples,assay of cellular function,exploration of cellular mechanisms and in vivo verification.Firstly,the expression characteristics of miR-127-3p in carotid atherosclerotic plaques were determined through assay of clinical samples.The expression and cellular localization of miR-127-3p in plaques of varying degree of development were determined by histological detection(immunohistochemistry,immunofluorescence,in situ hybridization,etc.),molecular biological technology(RT-q PCR)and serological detection,and the relationship between different pathological characteristics of plaques and the expression level of miR-127-3p was explored.Second,the in vitro effect of miR-127-3p on macrophages was determined.Based on the murine macrophages infected with lentivirus carrying miR-127-3p-overexpressed or-inhibited plasmid,Western bloting,RT-q PCR,flow cytometry,and lipid quantification,etc.were used to identify the macrophage function regulated by miR-127-3p.Third,the macrophages overexpressing or inhibiting miR-127-3p was used to explore the regulatory mechanisms of miR-127-3p on macrophage function and target molecules.The mechanisms of miR-127-3p regulating macrophage function were elucidated by RNA sequencing,targeted lipid metabonomics and quantification of lipid metabolites.Finally,the role of miR-127-3p in AS was further verified by in vivo experiments.Using Ldlr-/-mice and mice model of carotid unstable plaque injected with miR-127-3p agomir and antagomir,the role of miR-127-3p in the formation,progression and stability of carotid atherosclerotic plaques was determined by histological analysis and molecular biological techniques.ResultsPart Ⅰ:(1)Mi R-127-3p expression was significantly increased in carotid atherosclerotic plaque,and was even higher in unstable plaque(P<0.0001).(2)Mi R-127-3p was localized in lesional macrophages in carotid atherosclerotic plaque.(3)The expression of miR-127-3p was positively correlated with the degree of macrophage infiltration and intraplaque hemorrhage(P<0.0001).Part Ⅱ:(1)Overexpressing Mi R-127-3p promoted macrophage proliferation(P<0.05).(2)Overexpressing Mi R-127-3p promoted the accumulation of total cholesterol in macrophages(P<0.01)and aggravated foam cell formation(P<0.01).Part Ⅲ:(1)Stearoyl coenzyme A(SCD-1)was the target gene of miR-127-3p in macrophages and was negatively regulated by miR-127-3p(P<0.05).(2)Mi R-127-3p inhibited the synthesis of parts of unsaturated fatty acids by suppressing the expression of SCD-1(P<0.05),thereby increasing the content of acetyl coenzyme,substrate for cholesterol synthesis(P<0.001),finally causing an increase in cholesterol content(P<0.01)and foam cell formation.(3)Mi R-127-3p promoted macrophage proliferation by inhibiting SCD-1 expression(P<0.0001).Part Ⅳ:(1)In the Ldlr-/-mice group injected with miR-127-3p agomir,the plaque area was increased(P<0.001),necrotic core ratio increased(P<0.01)compared to controls;while in the Ldlr-/-mice group and mice of unstable plaque injected with miR-127-3p antagomir,the plaque area in carotid artery was decreased(P<0.05),necrotic core ratio decreased(P<0.05),and intraplaque hemorrhage was attenuated(P<0.05),thereby enhancing the stability of carotid plaque.(2)Mi R-127-3p negatively regulated the expression of SCD-1 in murine artery plaque(P<0.05).ConclusionmiR-127-3p is significantly upregulated in unstable carotid atherosclerotic plaques and expressed in lesional macrophages.The expression of miR-127-3p is positively associated with macrophage infiltration.Mi R-127-3p inhibits the expression of SCD-1 and blocks the synthesis of unsaturated fatty acids,thereby increasing the content of acetyl coenzyme A,which further enhances cholesterol accumulation in macrophage and aggravates foam cell formation.Those effects result in increased plaque area and the ratio of necrotic core,which finally lead to AS progression and decreased plaque stability.