微小RNA-19a对肝细胞LO2脂肪分解代谢的影响

发布时间：2019-03-06 20:59

【摘要】：目的:观察微小RNA-19a(microRNA-19a,miR-19a)对肝细胞LO2脂肪分解代谢的影响,并初步探索可能机制。方法:在肝细胞LO2中转染miR-19a模拟物(mimics)或miR-19a抑制物(inhibitor),用实时荧光定量PCR检测miR-19a的水平变化,利用生物信息学网站查找miR-19a的靶点,进而用双萤光素酶报告基因实验验证miR-19a对过氧化物酶体增殖物激活受体α(peroxisome proliferator-activated receptorα,PPARα)3’UTR活性的影响,通过Western blotting法检测PPARα及其下游脂肪分解代谢关键限速酶酰基辅酶A脱氢酶(acyl-coenzyme a dehydrogenase,ACADM)和肉毒碱棕榈酰基转移酶1A(carnitine palmitoyltransferase 1A,CPT1A)蛋白水平的变化,通过β-羟丁酸(beta-hydroxybutyric acid,β-OHB)试剂盒检测肝细胞LO2内酮体的生成能力。结果:转染miR-19a mimics可显著升高LO2细胞中的miR-19a水平(P0.05),而转染miR-19a inhibitor可明显抑制LO2细胞中的miR-19a水平(P0.05);生物信息学分析提示PPARα可能为miR-19a的潜在靶点,双萤光素酶报告基因实验证实了miR-19a mimics可使PPARα的3’UTR活性明显降低,miR-19a inhibitor可使PPARα的3’UTR活性显著升高,同时伴随PPARα及其下游基因ACADM和CPT1A的蛋白水平变化。此外,miR-19a mimics明显降低肝细胞LO2中β-OHB的含量(P0.05),miR-19a inhibitor可明显上调肝细胞LO2中β-OHB的含量(P0.05)。结论:miR-19a可通过调控PPARα及其下游关键限速酶的表达,调节肝细胞的脂肪分解代谢能力。
[Abstract]:Aim: to observe the effect of tiny RNA-19a (microRNA-19a,miR-19a) on lipid catabolism of LO2 in hepatocytes and explore the possible mechanism. Methods: the level of miR-19a was detected by real-time fluorescence quantitative PCR (real-time fluorescence quantitative PCR) after transfection of miR-19a analogue (mimics) or miR-19a inhibitor (inhibitor), in hepatocyte LO2. Bioinformatics website was used to find the target of miR-19a. The effects of miR-19a on the activity of peroxisome proliferator-activated receptor 伪 (peroxisome proliferator-activated receptor 伪, PPAR 伪) 3'UTR were examined by double luciferase reporter gene assay. The changes of protein levels of PPAR 伪 and acyl-coenzyme a dehydrogenase,ACADM and carnitine palmitoyl transferase 1A (carnitine palmitoyltransferase 1A, CPT 1A were detected by Western blotting method. 尾-hydroxybutyric acid (beta-hydroxybutyric acid, 尾-OHB) kit was used to detect the production of ketones in LO2. Results: transfection of miR-19a mimics could significantly increase the level of miR-19a in LO2 cells (P0.05), while transfection of miR-19a inhibitor could significantly inhibit the level of miR-19a in LO2 cells (P0.05). Bioinformatics analysis indicated that PPAR 伪 might be a potential target of miR-19a. Double luciferase reporter gene assay confirmed that miR-19a mimics could significantly decrease 3'UTR activity of PPAR 伪 and miR-19a inhibitor could significantly increase 3'UTR activity of PPAR 伪. At the same time, the protein levels of PPAR 伪 and its downstream genes ACADM and CPT1A were changed. In addition, miR-19a mimics significantly decreased the content of 尾-OHB in hepatocyte LO2 (P0.05), and miR-19a inhibitor significantly up-regulated the content of 尾-OHB in hepatocyte LO2 (P0.05). Conclusion: miR-19a can regulate the lipid catabolism ability of hepatocytes by regulating the expression of PPAR 伪 and its downstream key rate limiting enzymes.
【作者单位】：咸宁市中心医院内分泌科;湖北科技学院临床学院;湖北科技学院药学院;
【分类号】：R3416

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