炎症或非炎症条件下microRNA-101对细胞胆固醇外排影响的分子机制

发布时间：2018-02-23 22:07

本文关键词： miR-101 ABCA1 炎症因子　出处：《重庆医科大学》2015年博士论文　论文类型：学位论文

【摘要】：目的：探讨在炎症或非炎症条件下microRNA-101(miR-101)对人单核巨噬细胞(THP-1)和人肝癌细胞(HepG2)胆固醇外排的影响,为揭示miR-101在动脉粥样硬化发生中的作用机制提供实验依据。材料与方法：构建miR-101或Anti-miR-101慢病毒载体并建立高表达miR-101或Anti-miR-101细胞模型。使用双荧光素酶报告基因检测验证miR-101与三磷酸腺苷结合盒转运体Al(ABCAl) mRNA的3’非编码区(3’UTR)互补结合,分组为野生型(WT)+Con-miR组、定点突变(SDM)+miR-101组与WT+miR-101组。非炎症条件下THP-1与HepG2各分为对照组,miR-101组和Anti-miR-101组；炎症条件下各分为对照组,miR-101组,Anti-miR-101组,IL-6组(0.2%BSA+20ng/ml IL-6), TNF-a组(0.2%BSA+25ng/ml TNF-a), IL-6+miR-101/ Anti-miR-101组及TNF-a+Anti-miR-101组。各组再根据是否用LDL (0.2%BSA+25μg/ml LDL)处理,分为LDL负荷和无LDL负荷。采用oil red O染色检测细胞内脂质蓄积,酶法测定胞内胆固醇含量,荧光标记胆固醇法(BODIPY-cholesterol)检测载脂蛋白A-I(apoA-I)介导的胆固醇外排。运用实时荧光定量RT-PCR检测miR-101基因表达,Western Blotting检测ABCA1蛋白表达。结果：在结合位点2,与WT+Con-miR组和SDM+miR-101组比较,WT+miR-101组ABCA1 3'UTR活性显著降低,差异有统计学意义(P0.05)。非炎症状态,THP-1和HepG2在有无LDL负荷条件下,与对照组相比,miR-101组的ABCA1蛋白表达显著降低,apoA-I介导的细胞胆固醇外排明显减少,差异有统计学意义(P0.05)。Anti-miR-101组ABCA1蛋白表达明显增加,apoA-I介导的细胞胆固醇外排显著增加,差异有统计学意义(P0.05)。miR-101组或Anti-miR-101组分别可以促进或减少THP-1和HepG2胞内胆固醇聚集,差异有统计学意义(P0.05)。在炎症状态下,与对照组比较,IL-6组与TNF-a组HepG2miR-101表达显著增加,IL-6组THP-1 miR-101表达明显增加,差异有统计学意义(P0.05)。与IL-6组和TNF-α组比较,IL-6+Anti-miR-101组与TNF-a+Anti-miR-101组THP-1和HepG2 ABCA1蛋白表达明显增加,差异有统计学意义(P0.05)。结论：miR-101通过与ABCA1基因3’UTR端互补结合下调细胞ABCA1蛋白表达抑制胆固醇外排；炎症可以使细胞miR-101表达增加并增强miR-101促进胆固醇聚集的作用；下调细胞miR-101表达可以减轻炎症对ABCA1蛋白抑制作用。miR-101在动脉粥样硬化的发生发展过程中起到重要作用。
[Abstract]:Objective: to investigate the effects of microRNA-101 miR-101) on cholesterol efflux in human mononuclear macrophages (THP-1) and hepatocellular carcinoma cells (HepG2) under inflammatory or non-inflammatory conditions. Materials and methods: construction of miR-101 or Anti-miR-101 lentivirus vector and establishment of high expression miR-101 or Anti-miR-101 cell model. Double luciferase reporter gene detection test. The results show that miR-101 is complementary to the 3'noncoding region of the adenosine triphosphate binding cassette transporter (Alo ABCAl) mRNA. They were divided into two groups: wild type WTM Con-miR group, site-directed mutagenesis (SDM) miR-101 group and WT miR-101 group. Under non-inflammatory condition, THP-1 and HepG2 were divided into two groups: control group (n = 10) and control group (n = 10) and Anti-miR-101 group (n = 10). Under the inflammatory condition, each group was divided into two groups: control group, control group, anti-miR-101 group, anti-miR-101 group, IL-6 group, BSA 20 ng / ml IL-6, TNF-a group, 0.2 BSA25 ng / ml TNF-aI, IL-6 miR-101 / Anti-miR-101 group and TNF-a Anti-miR-101 group. Each group was divided into LDL load and no LDL load according to LDL 0.2BSA 25 渭 g / ml LDL. Oil red O staining was used to detect intracellular lipid accumulation. Enzymatic determination of intracellular cholesterol, The cholesterol efflux mediated by apolipoprotein A-Iapo A-I was detected by fluorescent cholesterol labeling method. The expression of ABCA1 protein was detected by real-time fluorescence quantitative RT-PCR. Results: at binding site 2, it was compared with WT Con-miR group and SDM miR-101 group. The activity of ABCA1 3 UTR was significantly decreased in WT miR-101 group. The difference was statistically significant (P 0.05). The expression of ABCA1 protein in THP 1 and HepG2 group was significantly lower than that in control group under the condition of LDL loading or not, and the cholesterol efflux mediated by apoA-I was significantly decreased in the control group. The expression of ABCA1 protein in P0.05 + Anti-miR-101 group was significantly increased, and the cholesterol efflux mediated by apoA-I was significantly increased. The difference was statistically significant (P 0.05). MiR-101 group or Anti-miR-101 group could promote or decrease the accumulation of intracellular cholesterol in THP-1 and HepG2, respectively. Under the condition of inflammation, the expression of HepG2miR-101 in IL-6 group and TNF-a group was significantly increased compared with that in control group. The expression of THP-1 miR-101 in IL-6 group was significantly higher than that in control group, and the expression of THP-1 miR-101 in IL-6 group was significantly higher than that in control group. Compared with IL-6 group and TNF- 伪 group, the expression of THP-1 and HepG2 ABCA1 in IL-6 Anti-miR-101 group and TNF-a Anti-miR-101 group were significantly increased. The difference was statistically significant (P 0.05). Conclusion: miR-101 can inhibit cholesterol efflux by complementarily binding with ABCA1 gene 3UTR, inflammation can increase the expression of miR-101 and enhance the effect of miR-101 on the accumulation of cholesterol. Down-regulation of miR-101 expression can reduce the inhibitory effect of inflammation on ABCA1 protein. MiR-101 plays an important role in the development of atherosclerosis.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R543.5

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