Bhatia H, Pattnaik B R, Datta M
CSIR-Institute of Genomics and Integrative Biology, Delhi, India.
Int J Obes (Lond). 2016 May;40(5):861-9. doi: 10.1038/ijo.2015.225. Epub 2015 Oct 26.
Hepatic expression of microRNA-107 is ubiquitously upregulated in various metabolic diseases. In our previous study, we had demonstrated that fatty acid synthase (FASN) is a target of miR-107. miR-107-FASN interaction, by inducing endoplasmic reticulum (ER) stress, promotes lipid accumulation in hepatocytes. Here, we explore the possible mechanism(s) of the miR-107-FASN-ER stress on hepatic lipid metabolism.
HepG2 cells were transfected with the scramble or miR-107 and/or its inhibitor. Transcript levels of lipid droplet membrane proteins, apolipoproteins and β-oxidation genes were quantified by quantitative reverse transcription-PCR. Cells were treated with tunicamycin (Tm, 1 h) and 4-PBA (4-phenyl butyric acid, 8 h) or transfected with hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, apha subunit (HADHA) short interfering RNA or its overexpression vector. Mice were injected with the scramble or mmu-miR-107 (2.5 mg kg(-1) body weight) and random glucose levels were measured and oral glucose tolerance test was performed. Serum levels of cholesterol, triglyceride and serum glutamic oxaloacetic transaminase/serum glutamic-pyruvic transaminase (SGOT/SGPT) were evaluated. Hepatic tissues were collected to estimate levels of miR-107 and mitochondrial β-oxidation genes. Six-micrometer-thick cryosections of hepatic tissues were prepared and stained with Oil Red O for lipid accumulation.
miR-107 does not alter the expression of lipid metabolism-related transcription factors, lipid droplet components and apolipiprotein B. miR-107 significantly decreased the levels of the mitochondrial β-oxidation enzyme, HADHA in HepG2 cells (P<0.01), which was prevented by the miR-107 inhibitor. Similar decrease was observed with Tm (P<0.001), suggesting that HADHA inhibition is promoted by ER stress induction. Interestingly, miR-107-mediated HADHA suppression was rescued by the ER stress inhibitor, 4-PBA (P<0.01). HADHA overexpression rescued miR-107-induced lipid accumulation (P<0.01). miR-107 injection in mice increased random blood glucose levels (P<0.05) and impaired glucose tolerance (P<0.05). Hepatic levels of Hadha were significantly decreased (P<0.001 and P<0.05) accompanied by increased lipid accumulation (P<0.001).
miR-107 promotes hepatic lipid accumulation by suppressing transcript levels of HADHA, induces hyperglycemia and impairs glucose tolerance. We conclude that miR-107 regulation of fatty acid oxidation is an important contributor toward hepatic lipid accumulation.
微小RNA-107（miR-107）在各种代谢性疾病中肝脏表达普遍上调。在我们之前的研究中，我们已经证明脂肪酸合酶（FASN）是miR-107的一个靶点。miR-107与FASN的相互作用通过诱导内质网（ER）应激，促进肝细胞脂质积累。在此，我们探讨miR-107 - FASN - ER应激对肝脏脂质代谢的可能机制。
用乱序序列或miR-107和/或其抑制剂转染HepG2细胞。通过定量逆转录-聚合酶链反应定量脂质滴膜蛋白、载脂蛋白和β-氧化基因的转录水平。细胞用衣霉素（Tm，1小时）和4-苯基丁酸（4-PBA，8小时）处理，或用羟酰基辅酶A脱氢酶/3-酮酰基辅酶A硫解酶/烯酰基辅酶A水合酶α亚基（HADHA）小干扰RNA或其过表达载体转染。给小鼠注射乱序序列或mmu-miR-107（2.5 mg kg⁻¹体重），测量随机血糖水平并进行口服葡萄糖耐量试验。评估血清胆固醇、甘油三酯和血清谷草转氨酶/谷丙转氨酶（SGOT/SGPT）水平。收集肝脏组织以估计miR-107和线粒体β-氧化基因的水平。制备肝脏组织的6微米厚冰冻切片，并用油红O染色以检测脂质积累。
miR-107不改变脂质代谢相关转录因子、脂质滴成分和载脂蛋白B的表达。miR-107显著降低HepG2细胞中线粒体β-氧化酶HADHA的水平（P<0.01），miR-107抑制剂可阻止这种降低。用Tm处理也观察到类似的降低（P<0.001），表明ER应激诱导促进了HADHA的抑制。有趣的是，ER应激抑制剂4-PBA可挽救miR-107介导的HADHA抑制（P<0.01）。HADHA过表达可挽救miR-107诱导的脂质积累（P<0.01）。给小鼠注射miR-107会增加随机血糖水平（P<0.05）并损害葡萄糖耐量（P<0.05）。肝脏中Hadha的水平显著降低（P<0.001和P<0.05），同时脂质积累增加（P<0.001）。
miR-107通过抑制HADHA的转录水平促进肝脏脂质积累，诱导高血糖并损害葡萄糖耐量。我们得出结论，miR-107对脂肪酸氧化的调节是肝脏脂质积累的一个重要因素。
