| Background and Objective: Atherosclerotic cardiovascular disease is the leadingcause of morbidity and mortality worldwide. A large amount of evidence supports a pivotalrole of inflammation and immune responses in all phases of atherosclerosis(AS), frominitiation of the fatty streak to final breakout of acute coronary syndromes (ACS).MicroRNAs (miRNAs) have been demonstrated to be associated with inflammation andimmune responses. miRNAs are18-22nucleotide long non-coding RNA molecules thatregulate gene expression posttranscriptionally by triggering either translation repression orRNA degradation. Among various miRNA, inflammation-related miRNAs-microRNA-155(miR-155) have been found to be involved in the atherogenesis.The etiology of AS is ascribed to various factors, one of which is the increase inapoptosis. The underlying molecular mechanisms of the increase in apoptosis remainlargely unknown. Macrophage apoptosis has been identified as a prominent feature ofadvanced atherosclerotic plaques. Pathological studies of advanced atherosclerotic lesionshave revealed a strong correlation between macrophage apoptosis and large necrotic cores,thought to promote plaque rupture and acute vascular events. Oxidized low-densitylipoprotein (OxLDL) is an important pathogenetic factor in atherosclerosis and a potentialinducer of cell apoptosis. Previous studies have demonstrated OxLDL-induced apoptosis ina variety of tissues and cells, including endothelial cells, smooth muscle cells (SMCs) andmacrophages. An extrinsic pathway mediated by the death receptor family, mainlyinvolving Fas/FasL signaling and down-stream caspase-3, has been defined as theunderlying mechanism in OxLDL-mediated apoptosis. Since macrophage apoptosis playsan important role in atherogenesis and plaque destabilization, modulation of cell death mayprovide significant protection against advanced atherosclerotic plaque rupture. Accumulating evidence indicates that the apoptotic machinery is regulated by miRNAs. Asa multi-functional miRNA, miR-155was previously shown to participate in the regulationof immunological responses and apoptotic pathways.The relationship between miR-155and the progression of atherosclerotic diseases isunclear. This study first aimed to investigate the association between miR-155and theseverity and extent of coronary stenotic lesions and atherosclerotic plaques stability.Moreover,given its crucial involvement in apoptotic pathways, we hypothesized thatmiR-155plays a role in oxLDL-induced macrophage apoptosis. Accordingly, the primaryaim of the current study was to address the specific role of miR-155in oxLDL-inducedmacrophage apoptosis and clarify the underlying mechanisms.Methods:(1) We measured the miR-155expression in both freshly isolated peripheralblood monouclear cell (PBMCs) and plasma by real-time PCR in110consecutive patientsundergoing coronary computed tomography angiography (CTA) and coronary angiographyfor suspected coronary heart disease (CHD). The severity and extent of coronary stenoticlesions were evaluated on coronary angiography findings by Gensini score and the plaquesstability was evaluated on coronary CTA.(2) qRT-PCR was performed to detect miR-155expression in oxLDL-treated RAW264.7cells.(3) miR-155mimics, miR-155inhibitor,andnegative control were transiently transfected into RAW264.7cells or HEK-293cells to up-or down-regulate the miR-155expression level. Or the fas-associated deathdomain-containing protein (FADD)-expressing vector was used to infect RAW264.7cellsto up-regulate the FADD expression level.(4)Cell viability assays (Cell Counting Kit-8)was performed in oxLDL-treated RAW264.7cells,with or without transfection of miR-155mimics/inhibitor.(5) TUNEL assay, Annexin V/propidium iodide (PI) staining,andcleaved caspase-3(17kDa) protein expression were used to assess the macrophageapoptosis.(6) Bioinformatics technique was used to predict the potential target of miR-155.(7) Luciferase assays was performed to validate the putative miR-155target.(8) Westernblotting (WB) was used to detect caspase-3and FADD protein expression.Results:(1)The miR-155expression was significantly lower in56patients with CHDthan those in54controls (p <0.01).(2) The level of miR-155in PBMCs or plasma waslower in patients with unstable angina pectoris (UAP,n=31) and acute myocardial infarction(AMI,n=24) than in patients with chest pain syndrome (CPS,n=31), while no statistically significant differences were observed between patients with stable angina pectoris(SAP,n=24) and CPS.(3) Spearman’s correlation analysis showed that the expression ofmiR-155in plasma positively correlated with the expression in PBMCs.(4) The levels ofmiR-155in the subjects with diseased vessels of2and>=3were significantly lower thanthose with diseased vessel of0and1. The levels of miR-155were not significantlydifferent among groups with diseased vessels of0and1.(5) miR-155were negativelyassociated with Gensini scores(r=-0.663,p<0.001).(6) The miR-155level in patiens of softplaque is significantly lower than those with calcified plaque and fibrous plaque.(7) ThemiR-155expression was significantly correlated to age (r=-0.227), hypertension (r=-0.440),total cholesterol (r=0.239), high-density lipoprotein (HDL) cholesterol (r=0.280),low-density lipoprotein(LDL) cholesterol (r=-0.315), tobacco (r=-0.363),ACEI (r=-0.250),statins(r=-0.368)，high sensitivity C-reactive protein (hs-CRP)(r=-0.515).(8) Constitutiveexpression of miR-155in RAW264.7cells was increased following stimulation withOxLDL in a dose-and time-dependent manner.(9) OxLDL-treated RAW264.7cells showeda marked increase in apoptosis, which was suppressed in the presence of miR-155mimicsand increased with antagonists of miR-155.(10) Bioinformatics analysis revealed FADD asa putative target of miR-155.(11) Luciferase reporter assay and WB further disclosed thatmiR-155inhibits FADD expression by directly targeting the3’-UTR region.(12) miR-155inhibits oxLDL-induced apoptosis, but overexpression of FADD limits this anti-apoptoticactivity.Conclusion: miR-155expression is inversely associated with complicatedproatherogenic metabolic risk factors, and the severity of coronary stenotic lesionscalculated by Gensini scores, supporting a protective role for miR-155against theprogression of atherosclerosis. Moreover,our results collectively suggest that miR-155attenuates apoptosis of OxLDL-mediated RAW264.7cells by targeting FADD, supporting apossible therapeutic role in atherosclerosis. |
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