| BackgroundMicroRNAs are small regulatory RNA molecules encoded in plant and animal genomes that regulate the expression of target genes by binding to the3'un-translated regions of specific mRNAs, thereby triggering mRNA degradation or translational repression. Of the regulatory RNA classes, microRNAs have been shown to play an important role in lipid metabolism and in cells related in atherosclerosis. Atherosclerosis is a complex process characterized by lipid accumulation in the arterial wall. Atherogenesis starts with the attraction, recruitment, and activation of different cell types, including monocytes/macrophages, T lymphocytes, endothelial cells, intimal smooth muscle cells, and adipocytes. This cellular activation provokes a local inflammatory response. Low-density lipoprotein cholesterol is one of the most important risk factors in atherosclerosis, and oxidized low-density lipoprotein is involved in the atherosclerotic procedure. Until now, few studies are reported to be focused on the relationship between oxidized low-density lipoproteins and the expression of miRNA-143in3T3-L1adipocytes.Atherosclerotic cardiovascular disease is a major health problem around the world. Obesity is a primary risk factor for atherosclerosis and is associated with increased morbidity and mortality of cardiovascular diseases. However, the precise molecular pathways underlying this close association remain poorly understood. Adipokines are cytokines, chemokines and hormones secreted by adipose tissue that couple the regulation of lipid accumulation, inflammation, and atherogenesis, and therefore serve to link obesity with cardiovascular disorders. Adiponectin, a30-kDa adipose-specific secreted and inflammation-related protein, is now a recognized component of a important signaling network among adipocytes, which has been found is a protective factor for insulin-resistance, diabetes and atherosclerosis. The protective effect of adiponectin has been attributed to its anti-inflammatory action. MicroRNAs are a class of small endogenous non-coding RNAs, which function as important regulators of a wide range of cellular processes by modulating gene expression. Recent studies have shown that dysregulation of miRNA expression is closely associated with many diseases, including obesity, type2diabetes and atherosclerosis. However, the possible role of microRNAs in the secretion of adiponectin in adipocyte remains to be determined.Despite advances in the prevention and management of cardiovascular disease (CVD), this multi-factorial disorder remains a leading cause of mortality worldwide. Peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptor α (LXRα) are nuclear receptors that play pivotal roles in adipocyte cholesterol homeostasis and inflammation; key biological processes in atherogenesis. The activation of PPARy and LXRa by natural or synthetic ligands results in the trans-activation of ABCA1, which is one of the integral players in cholesterol efflux and reverse cholesterol transport. PPARy-LXRa pathway plays an important role in adipocytes cholesterol efflux. Lipid metabolism is tightly regulated at the cellular level. In addition to classic transcriptional regulation of cholesterol metabolism mentioned above, members of a class of non-coding RNAs termed microRNAs have now been identified to be potent posttranscriptional regulators of lipid metabolism genes involved in cholesterol homeostasis and fatty acid oxidation. Several reports have recently shown that miR-33regulates cholesterol efflux and HDL biogenesis by down-regulating the expression of the ABC transporters. Other microRNAs including miR-122, miR-370, miR-335, and miR-378/378*, miR-27and miR-125a-5p have been implicated in regulating cholesterol homeostasis, fatty acid metabolism and lipogenesis. Nevertheless, few researchers focused on the relationship between miRNA-143and its effect on the cholesterol efflux. The specific role of miRNA-143in regulating lipid metabolism is a burgeoning area of investigation.ObjectiveAlthough the role for microRNAs (miRNAs) in regulating multiple physiological processes including apoptosis, cell differentiation, and cancer is well established, the importance of these tiny RNAs in regulating lipid metabolism especially in atherosclerosis has only recently been uncovered. The aim of this study was to evaluate the expression of miRNA-143in fully differentiated3T3-L1adipocytes with the stimulation of oxidized low-density lipoproteins, and to explore the effect of both miRNA-143mimics and miRNA-143inhibitor on the secretion and the expression of adiponectin and adipocyte cholesterol efflux, and the underlying possible mechanismMethodsAdipocytes were divided into different groups in order to be incubated in the medium containing various concentrations of ox-LDL (0,50,100mg/ml) and to be incubated for various periods of time (6,12,24h). Evaluate the levels of miRNA-143by real-time PCR in adipocytes and explore the relationship between both of them. Adipocytes were incubated and then were transfected by miRNA-143mimics or inhibitor in various concentrations (0-100nM). Evaluate the levels of adiponectin, the mRNA and protein expression of adiponectin and PPARγ.Adipocytes were labeled and then were transfected with various concentration of miRNA-143mimics or inhibitor. We determined the rate of adipocytes cholesterol efflux mediated by extracellular acceptor apoAI via liquid scintillation counting. We detected the mRNA and protein expressions of ABCA1, LXRα and PPARγ in adipocytes by RT-PCR or western blot.Results1. Mouse fully differentiated adipocytes express miRNA-143.2. After treated with different concentration of ox-LDL (10ug/ml,20ug/ml), the expression of miRNA-143was decreased (0.61±0.12,0.40±0.09; p<0.05). Ox-LDL induced miRNA-143expression in a concentration-dependent manner.3. After treated with ox-LDL for different periods of time(6h,12h,24h), the expression of miRNA-143in adipocytes were decreased (0.87±0.10,0.63±0.12,0.392±0.06; p<0.05).Ox-LDL induced miRNA-143expression in a time-dependent manner.4. Full differentiated3T3-L1adipocytes that transfected with miRNA-143mimics would be induced to up-regulate the expression of both mRNA and protein of adiponectin and PPARy (145.5±7.20,175.1±2.25;196.3±3.14,230.0±3.23; p<0.01). Those adipocytes transfected with miRNA-143inhibitor down-regulated the expression of both mRNA and protein of adiponectin and PPARy (46.3±0.80,40.5±0.62;32.4±0.82,29.1±1.25; p<0.01).5. MiRNA-143induced the mRNA and protein expression of adiponectin and PPARy in a concentration-dependent manner. Compared with negative control group, various concentrations of miRNA-143mimics (50nM,100nM) increased the mRNA expression of adiponectin (143.3±2.55,187.8±15.03), and increased its protein expression (139.1±3.34,154.3±3.34).6.Various concentrations of miRNA-143inhibitors (50nM,100nM) decreased the mRNA expression of adiponectin (70.8±2.09,43.1±2.45), and decreased its protein expression (65.6±3.91,30.0±0.65). PPARy pathway may participate in the process mentioned above.7. MiRNA-143transfection would influence cholesterol efflux rate in fully differentiated3T3-L1adipocytes. Treated with different concentrations of miRNA-143mimics(0nM,50nM,100nM), the adipocytes cholesterol efflux mediated by apoAI was dose-dependently increased, while the miRNA-143inhibitor dose-dependently decreased the adipocytes cholesterol efflux rate.8. The mRNA expression of PPARγ, LXR-α, ABCA1were influenced after the adipocytes transfected with miRNA-143. MiRNA-143mimics would increase the expression to175.1±2.25,175.8±11.58,188.6±5.16, while miRNA-143inhibitor would decrease their expression to40.5±0.62,35.8±2.20,40.5±0.42.(p<0.05).9. The protein expression of PPARγ, LXR-α, ABCA1were also influenced after the adipocytes transfected with miRNA-143. Compare with the mock group, miRNA-143mimics would increase the expression to230.0±3.23,228.9±6.63,199.6±3.85, while miRNA-143inhibitor would decrease their expression to29.1±1.25,32.7±1.77,37.0±0.70.(p<0.05)Conclusion1. Fully differentiated3T3-L1adipocytes express miRNA-143. 2. Ox-LDL induced miRNA-143expression in3T3-L1adipocytes in a concentration-dependent and time-dependent manner.3. MiRNA-143transfection influences the secretion of adiponectin in adipocytes.4. miRNA-143mimics could concentration-dependently up-regulate the mRNA and protein expression of PPARy and adiponectin in3T3-L1adipocytes, while miRNA-143inhibitor would down-regulate the expression of both of them in a dose-dependent manner.5. PPARγ pathway may participate in the process of miRNA-143regulating the secretion of adiponectin.6. MiRNA-143significantly influences the rate of adipocytes cholesterol efflux.7. Treated with miRNA-143mimics, the mRNA and protein expression of PPARy, LXR-α, ABCA1in adipocytes were up-regulated, while miRNA-143inhibitor transfection have shown opposite effect.8. The effects of miRNA-143on PPARy-LXRa pathway could influence the mRNA and protein expressions of ABCA1in adipocytes, which contributed to the adipocytes cholesterol efflux rate.
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