Clinical Significance Of MiR-146a-5p In Unstable Angina Pectoris And Its Inflammatory Inhibition Roles In Cholesterol Metabolism

Part 1: Clinical Significance of Circulating Levels of mi R-146a-5p inPlasma of Patients with Unstable Angina PectorisObjective: We examined circulating levels of miR-146a-5p in plasma of patients with unstable angina pectoris(UA) in order to discover potential biomarkers for the diagnosis and prognosis of UA patients.Methods: Circulating levels of miR-146a-5p were examined by TaqMan real-time PCR in the plasma of troponin-negative UA(n=23) and Sex-and Age-matched control subjects(n=28). Receiver Operating Characteristic(ROC) curves were calculated to estimate the area under the curve(AUC) for circulating levels of miR-146a-5p in UA patients.Results: Circulating levels in plasma from UA patients were significantly reduced(P<0.0001), compared with control subjects. To further investigate the capacity of miR-146a-5p as a potential biomarker, we performed the ROC curve analyses. Mi R-146a-5p distinguishes UA patients from control subjects with an area under the curve(AUC) of 0.997(95% CI, 0.9886 to 1.005, P<0.0001).Conclusions: This study showed that circulating levels of mi R-146a-5p were significantly reduced in UA patients. ROC analysis suggest circulating level of miR-146a-5p may be a potential biomarker for diagnosis and prognosis of UA patients. Taken together, circualting miR-146a-5p may be associated with the pathogenesis of atherosclerosis.Part 2: AGEs and P.g-LPS can Upregulate the Expression of miR-146a-5p in PMA-induced THP-1 macrophagesObjective: Several researches have confirmed that mi R-146 a playimportant roles in innate and adaptive immune responses by anti-inflammatory mechanism. Previous studies showed some endogenous and exogenous inflammatory factors like advanced glycation end-products(AGEs) and P.gingivalis lipopolysaccharide(P.g-LPS) are independenet risk factors for atherosclerotic diseases. However, the interaction mechansim between these infammatory risk factors and atherosclerotic diseases remains unclear. This study aimed to explore whether the inflammatory factors of endogenous AGEs and exogenous P.g-LPS regulate the expression of miR-146a-5p in macrophages in vitro or not.Methods: The expression of miR-146a-5p in PMA-induced THP-1macrophages stimulated by P.g-LPS or AGE-BSA were detected by TaqMan real-time PCR.Results: The expression levels of miR-146a-5p in LPS or AGE-BSA group were almost 9(P< 0.01) and 2.3(P< 0.05) times higher than in control group, respectively.Conclusions: Both endogenous inflammatory factor AGEs and exogenous P.g-LPS can upregulate the expression of mi R-146a-5p in PMA-induced THP-1 macrophages.Part 3: Effects of miR-146a-5p in Attenuating P.g-LPS- and AGEs-induced ABCA1 and ABCG1 ExpressionObjective: Several researches have confirmed that miR-146 a play important roles in innate and adaptive immune responses by anti-inflammatory mechanism. These inflammatory responses induced by endogenous and exogenous inflammatory factors usually result in the changes of ATP-binding cassette transporter A-1(ABCA1) or ATP-binding membrane cassette transporter G-1(ABCG1), involved in cholesterol metabolism. However, the roles of mi R-146 a in regulating cholesterol metabolism induced by inflammatory factors remain unclear. The goal of this study is to determine the effects of miR-146a-5p on LPS- and AGE-BSA-induced ABCA1 and ABCG1 pathway.Methods: miR-146a-5p mimics or control mimics were transfected into PMA-induced THP-1 macrophages using in vitro transiently transfected technology. After 24 h of transfection, these macrophages were stimulated with P.g LPS(1μg/mL) or AGE-BSA(100 μg/mL) for another 24 h. Protein levels of ABCA1 and ABCG1 in THP-1 macrophages were detected by Western blotting.Results: Protein levels of ABCA1 and ABCG1 in mi R-146a-5p transfected group were not significantly changes(P> 0.05), compared with control group. whereas protein levels of ABCA1 and ABCG1 in P.g LPS- or AGE-BSA-stimulated THP-1 macrophages were significantly higher or lower than in control group(P < 0.05), respectively. Protein levels of ABCA1 and ABCG1 in the group costimulated with mi R-146a-5p and P.g LPS were significantly lower than in LPS group(P< 0.05). Meanwhile, those in the group costimulated with miR-146a-5p and AGE-BSA were significantly higher than in AGE-BSA group(P< 0.05).Conclusions: Our findings indicate the effect of exogenous inflammatory factor P.g LPS in regulating protein levels of ABCA1 and ABCG1 in macrophages are opposite to that of endogenous AGEs. miR-146a-5p can attenuate their effect in regulating protein levels of ABCA1 and ABCG1,athough it do not directly involved in regulating cholesterol metabolism in macrophages under the condition of non-inflammatory stimulation.Part 4: Possible Mechanism of the Effects of mi R-146a-5p in AttenuatingP.g-LPS-and AGEs-induced ABCA1 and ABCG1 ExpressionObjective: Our studies have showed miR-146a-5p can attenuate the effect of ABCA1 and ABCG1 induced by P.g LPS and AGE-BSA. The purpose of this study is to explore whether the inhibitory roles of miR-146a-5p are involved in regulating the expession of interleukin-1 receptor-associated kinases-1(IRAK-1) and key regulators of cholesterol metabolism, liver X receptors(LXRs).Methods: miR-146a-5p mimics or control mimics were transfected intoPMA-induced THP-1 macrophages using in vitro transiently transfected technology. After 24 h of transfection, these macrophages were stimulated with P.g LPS(1μg/mL) or AGE-BSA(100 μg/mL) for another 24 h. Protein levels of IRAK-1 and LXRα/β in THP-1 macrophages were detected by Western blottingResults: Protein levels of IRAK-1 in THP-1 macrophages after 48 h of mi R-146a-5p transfection were significantly decreased(P< 0.05), compared with control group, whereas those in LPS and AGE-BSA group were significantly higher than in control group(P< 0.05), respectively. Protein levels of IRAK-1 in costimulated group with miR-146a-5p and P.g LPS were significantly lower than in LPS and AGE-BSA group(P< 0.05). In contrast to their effect in regulating IRAK-1 expression, miR-146a-5p, P.g LPS and AGEs do not change protein levels of LXRα/β(all P> 0.05).Conclusions: Our findings suggest the effects of miR-146a-5p in attenuating P.g LPS- and AGEs-induced ABCA1 and ABCG1 expression are involved in target inhibitory regulation of IRAK-1, but not in the regulation of LXRα/β.