Mechanism Of Action Of MicroRNA-216a In Atherosclerotic Plaque Stability

Background:Atherosclerosis is an age-related,chronic inflammatory disorder.The unstable plaques lead to high morbidity and mortality of cardiovascular diseases such as myocardial infarction and stroke.Endothelial cell senescence has been involved in endothelial dysfunction and inflammation which promotes the process of atherogenesis.The monocyte/macrophages have been well-recognized as the central participant in the inflammatory process during atherosclerosis development,from initiation of lesions and lesion expansion to necrosis and eventually the rupture of plaques.Increasing evidence suggest a significant role of microRNAs(miRNAs)as mediators in vascular aging and atherosclerosis,however,the regulatory mechanisms of miRNAs in atherosclerotic plaque stability and clinical applications has not been fully clarified.We identified that microRNA-216a(miR-216a)significantly increased in senescent endothelial cells.In the present study,we would explore the regulatory role of miR-216a in plaque stability from the following three aspects:1.The replicative senescence model of human umbilical vein endothelial cells(HUVECs)and coronary artery disease(CAD)case control populations were established to explore the role of miR-216a in endothelial aging and dysfunction;2.To elucidate the molecular mechanisms of miR-216a on macrophages polarization and atherosclerotic plaque stability,the THP-1 macrophages model,animal model and CAD cohort were applied.3.We further aimed to find a small molecule drug that putatively binds to miR-216a and block its atherogenic effect.Methods and results:The first part indicated that Smad3 and Smad7 were the direct targets of miR-216a.Stable overexpression of miR-216a induced a premature senescence-like phenotype in HUVECs with an impairment in proliferation and migration,and led to an increased adhesion to monocytes.For molecular mechanisms,on the one hand,miR-216a inhibited Smad3 expression and thereafter modulated the expression of NF-?B inhibitor alpha(I?B?)and adhesion molecules;conversely,inhibition of endogenous miR-216a in senescent HUVECs rescued Smad3 and I?B?expression and inhibited monocytes attachment.On the other hand,blocking Smad7 with small interfere RNAs(siRNAs)was found to activate the NF-?B signaling by promoting the phosphorylation and degradation of I?B?,and thereafter promoted endothelial inflammation;when endogenous miR-216a was suppressed by its inhibitor,the Smad7/I?B? expression was rescued,which led to decrease endothelial inflammation and monocytes recruitment;moreover,in human carotid atherosclerotic plaques from patients who underwent carotid endarterectomy(n=41),Smad7 expression was markedly decreased by 30%(P=0.02)in high miR-216a group compared with low miR-216a group.Furthermore,the plasma miR-216a was significantly higher in old CAD patients(>50 years)and associated with increased 31%risk for CAD(odds ratio 1.31,95%confidence interval 1.03-1.66;P=0.03)compared with healthy controls(>50 years).The results of second part verified that Telomerase reverse transcriptase(TERT)was highly expressed in macrophages of human carotid atherosclerotic plaques.miR-216a was found to promote telomerase activation in macrophages by 4.5-fold(P=0.002)through the Smad3/NF-?B pathway.miR-216a also induced macrophages senescence characterized by senescence-associated-?-galactosidase activity and p53 and p16 expression.TERT overexpression promoted the transformation of M2 to M1 while this conversion was suppressed once TERT was inhibited,and the related inflammatory factors and lipid uptake ability of M1 cells were also increased by TERT.In the carotid plaques from miR-216a-treated apolipoprotein E-/-mice,the numbers of M1 macrophages were increased whereas M2 cells reduced,accompanying with inhibited Smad3 expression and upregulated inflammatory markers and TERT activity.Furthermore,plasma miR-216a level was higher in patients with vulnerable mixed plaques(n=181)than those with calcified plaques(n=73)and controls(n=264).In the third part,we introduced an artificial-intelligence-based tool,drug-RNA interaction predictor(DRIP)to predict the potential binding of ginsenoside Rb2 and miR216a.The Microscale Thermophoresis(MST)confirmed that Rb2 directly bound to miR216a in vitro;next,the experiments of luciferase reports in HEK293T cells showed that intracellular Rb2 can block the inhibition of miR-216a on Smad3 by competitive binding to miR-216a;additionally,we found Rb2 promoted the degradation of miR-216a in THP1 macrophages.Furthermore,our results showed that Rb2 partially rescued Smad3 protein expression in macrophages with miR-216a overexpression and inhibited miR216a-mediated macrophage senescence and lipid uptake ability.Conclusions:Taken together,our data suggested that miR-216a induced endothelial senescence and inflammation as an endogenous inhibitor of Smad3/Smad7/I?B? pathway and promoted M1 macrophages polarization and atherosclerosis progression by activating telomerase via the Smad3/NF-?B pathway.Moreover,the small molecule drug Rb2 inhibited miR-216a-mediated atherogenic effects including macrophages senescence and phagocytosis by directly binding to miR-216a.