| 韩 雪,董宝洁,柯 月,李梦莹,白 洁,纪文静.miR-206调控SIRT1/AMPK通路影响脂质代谢改善非酒精性脂肪肝的研究[J].,2024,(16):3032-3038 |
| miR-206调控SIRT1/AMPK通路影响脂质代谢改善非酒精性脂肪肝的研究 |
| MiR-206 Regulates Lipid Metabolism to Improve non-Alcoholic Fatty Liver Disease by The SIRT1/AMPK Pathway |
| 投稿时间：2023-12-12 修订日期：2023-12-31 |
| DOI：10.13241/j.cnki.pmb.2024.16.006 |
| 中文关键词: 非酒精性脂肪肝 miR-206 沉默信息调节因子1 |
| 英文关键词: Non alcoholic fatty liver disease MiR-206 Silent Information Regulatory Factor 1 |
| 基金项目:新疆神经系统疾病研究重点实验室开放课题项目(XJDX1711-2242)；新疆维吾尔自治区自然科学基金项目(2021D01C355) |
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| 中文摘要: |
| 摘要 目的：探讨miR-206对长链游离脂肪酸（FFA）诱导HepG2细胞建立的非酒精性脂肪肝（NAFLD）细胞模型的影响及其对沉默信息调节因子1（SIRT1）/AMPK通路的调控。方法：用1mM的FFA处理HepG2细胞24 h构建NAFLD细胞模型，根据转染物质的不同分为6组：Control组（无任何处理）、FFA组（构建NAFLD模型）、FFA+mimics NC组（转染mimics NC质粒）、FFA+miR-206 mimics组（转染miR-206 mimics质粒）、FFA+Vector组（转染Vector质粒）、FFA+OE-SIRT1组（转染OE-SIRT1质粒）。油红O染色法检测细胞中脂质蓄积情况，酶联免疫吸附试验（ELISA）方法检测谷草转氨酶（AST）、丙氨酸氨基转移酶（ALT）及甘油三酯（TG）含量，荧光定量聚合酶链反应（qRT-PCR）检测miR-206、SIRT1 mRNA表达量，Western blot法检测SIRT1、固醇调节元件结合蛋白1（SREBP1）、脂肪酸合成酶（FAS）、硬脂酰辅酶Al（SCD1）、乙酰CoA羧化酶1（ACC1）、白细胞分化抗原36（CD36）、AMP活化蛋白激酶（AMPK）及p-AMPK蛋白水平，TargetScan在线网站预测miR-206与SIRT1结合位点，双荧光素酶报告基因实验验证miR-206与SIRT1的靶向关系。结果：与Control组比较，FFA组细胞AST、ALT及TG含量明显升高，TG含量升高，miR-206、SIRT1蛋白及基因mRNA含量明显降低，SREBP1、FAS、SCD1、ACC1及CD36蛋白含量升高，p-AMPK/AMPK比值降低，差异具有统计学意义（P均＜0.01）。与FFA+mimics NC组细胞比较，FFA+miR-206 mimics组细胞内TG含量下降，SREBP1、FAS、SCD1、ACC1及CD36蛋白水平降低，SIRT1蛋白及基因mRNA水平升高，p-AMPK/AMPK比值升高，差异具有统计学意义（P均＜0.01）。与FFA+Vector组细胞比较，FFA+OE-SIRT1组细胞内TG含量下降，SREBP1、FAS、SCD1、ACC1及CD36蛋白水平降低，p-AMPK/AMPK比值升高，差异具有统计学意义（P均＜0.01）。TargetScan在线网站预测发现，miR-206与SIRT1野生型存在结合位点。SIRT1 WT+miR-206 mimics组细胞荧光素酶活性高于SIRT1 WT+mimics NC组，差异具有统计学意义（P＜0.01）。结论：上调miR-206能够通过SIRT1/AMPK通路减轻NAFLD引起的肝细胞脂质代谢。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effect of miR-206 on the non alcoholic fatty liver (NAFLD) cell model induced by long chain free fatty acids (FFA) in HepG2 cells and its relationship with silent information regulatory factor 1 (SIRT1)/AMPK pathway. Methods: HepG2 cells were treated with 1mM FFA for 24 hours to construct a NAFLD cell model. According to the different transfected substances, they were divided into 6 groups: Control group (without any treatment), FFA group (NAFLD model), FFA+mimics NC group (transfect mimics NC plasmid), FFA+miR-206 mimics group (transfect miR-206 mimics plasmid), FFA+Vector group (transfect Vector plasmid), and FFA+OE-SIRT1 group (transfect OE-SIRT1 plasmid). Oil red O staining was used to detect lipid accumulation in cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triglycerides (TG). Fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-206 and SIRT1 mRNA. Western blot was used to detect SIRT1, sterol regulatory element binding protein 1 (SREBP1), fatty acid synthase (FAS), stearoyl coenzyme Al (SCD1), acetyl CoA carboxylase 1 (ACC1), leukocyte differentiation antigen 36 (CD36), AMP activated protein kinase (AMPK), and p-AMPK protein levels. TargetScan was used to predict the binding site of miR-206 to SIRT1. Dual luciferase reporter gene experiments was used to validate the targeting relationship between miR-206 and SIRT1. Results: Compared with the Control group, the FFA group showed a significant increase in AST, ALT, and TG content, an increase in TG content, a significant decrease in miR-206, SIRT1 protein and gene mRNA content, an increase in SREBP1, FAS, SCD1, ACC1, and CD36 protein content, and a decrease in p-AMPK/AMPK ratio(P<0.01). Compared with the FFA+mimics NC group cells, the FFA+miR-206 mimics group cells showed a decrease in intracellular TG content, a decrease in SREBP1, FAS, SCD1, ACC1, and CD36 protein levels, an increase in SIRT1 protein and gene mRNA levels, and an increase in p-AMPK/AMPK ratio, with statistically significant differences (P<0.01). Compared with the FFA+Vector group cells, the FFA+OE-SIRT1 group cells showed a decrease in TG content, a decrease in SREBP1, FAS, SCD1, ACC1, and CD36 protein levels, and an increase in p-AMPK/AMPK ratio (P<0.01). There was a binding site between miR-206 and the SIRT1 wild-type. The cell luciferase activity in the SIRT1 WT+miR-206 mimics group was higher than that in the SIRT1 WT+mimics NC group(P<0.01). Conclusion: Upregulation of miR-206 can alleviate NAFLD induced lipid metabolism in liver cells through the SIRT1/AMPK pathway. |
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