miR-129-5p对脂肪细胞增殖、分化及棕色化的作用及机制研究

发布时间：2018-04-30 02:11

本文选题：miR-129-5p + 脂肪细胞　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：MicroRNA在脂肪的各个进程中都发挥着重要作用,包括脂肪细胞的增殖、分化以及棕色化。MiR-129-5p是一个重要的肿瘤抑制因子,它对很多癌细胞的增殖具有抑制作用。中南大学关于mi RNA的微阵列分析表明miR-129-5p在胰岛素抵抗的3T3细胞中高表达,预示着mi R-129-5p可能在脂肪细胞中发挥作用。本文旨在探索miR-129-5p在脂肪细胞增殖、分化及棕色化过程中发挥的作用。本文使用miR-129-5p mimic转染3T3细胞系和小鼠原代皮下脂肪细胞,并使用EdU、流式细胞术、CCK-8、油红O染色、Bodipy染色、RT-qPCR、Western blot等技术检测过表达miR-129-5p对脂肪细胞增殖、分化及棕色化的影响。同时构建了miR-129-5p靶基因G3BP1的荧光素酶报告载体,对miR-129-5p调控3T3增殖的机制进行探索。获得的主要结果如下:1.在人、小鼠、大鼠等物种间miR-129-5p的成熟序列及种子序列一致;miR-129-5p在小鼠脑、皮下白色脂肪、棕色脂肪中表达较高,且其表达水平在脂肪细胞增殖期间呈下降趋势,在分化期间呈升高趋势。2.过表达miR-129-5p可以影响cyclin D及p27等细胞周期相关基因的mRNA及蛋白水平表达;miR-129-5p显著减少了处于细胞周期S期的细胞数目同时显著增加了处于细胞周期G2期的细胞数目并造成G2期阻滞;过表达miR-129-5p在分子水平和细胞水平上显著抑制了3T3前体脂肪细胞的增殖过程。3.过表达miR-129-5p显著抑制了G3BP1基因mRNA及蛋白水平的表达;mi R-129-5p直接靶定G3BP1基因的3'UTR区域;过表达miR-129-5p显著升高了p38的蛋白水平及磷酸化水平表达;miR-129-5p是通过靶定G3BP1并影响下游p38信号通路发挥其抑制3T3前体脂肪细胞增殖作用的。4.过表达miR-129-5p后成脂相关基因PPARγ、CEBPα及aP2的mRNA及蛋白水平的表达没有发生变化;mi R-129-5p没有影响脂滴的形成及数量;mi R-129-5p过表达不影响脂肪细胞分化过程。5.罗格列酮法诱导小鼠脂肪细胞棕色化后棕色脂肪标志基因UCP1及Cidea的表达明显升高。过表达miR-129-5p使棕色脂肪标志基因UCP及PGC1-α的mRNA和蛋白水平表达显著降低;过表达miR-129-5p在分子水平上抑制了脂肪细胞棕色化过程。综上所述,mi R-129-5p可以通过靶定G3BP1并影响p38通路抑制3T3前体脂肪细胞的增殖过程,同时miR-129-5p可以在分子水平上抑制脂肪细胞的棕色化过程,但对脂肪细胞的分化过程没有影响。该研究结果为脂肪沉积性状的选育以及miRNA对白色脂肪棕色化的调控研究提供了理论依据。
[Abstract]:MicroRNA plays an important role in all processes of fat, including adipocyte proliferation, differentiation and browning. MiR-129-5p is an important tumor suppressor, which can inhibit the proliferation of many cancer cells. The microarray analysis of mi RNA from Central South University showed that miR-129-5p was highly expressed in insulin-resistant 3T3 cells, suggesting that mi R-129-5p might play a role in adipocytes. This paper aims to explore the role of miR-129-5p in adipocyte proliferation, differentiation and browning. In this paper, miR-129-5p mimic was used to transfect 3T3 cell line and primary subcutaneous adipocytes of mice. The effects of overexpression of miR-129-5p on proliferation, differentiation and browning of adipocytes were detected by using Edu, flow cytometry and oil red O staining. The luciferase report vector of miR-129-5p target gene G3BP1 was constructed to explore the mechanism of 3T3 proliferation regulated by miR-129-5p. The main results are as follows: 1. The mature sequence and seed sequence of miR-129-5p in human, mouse and rat were identical. The expression of miR-129-5p was higher in mouse brain, subcutaneous white fat and brown fat, and the expression level of miR-129-5p decreased during the proliferation of adipocytes. During the differentiation period, there was an increasing trend. 2. Overexpression of miR-129-5p could affect the expression of mRNA and protein of cell cycle related genes such as cyclin D and p27. MiR-129-5p significantly reduced the number of cells in S phase of cell cycle, and increased the number of cells in G2 phase of cell cycle and resulted in arrest of G2 phase. Overexpression of miR-129-5p significantly inhibited the proliferation of 3T3 precursor adipocytes at molecular and cellular levels. Overexpression of miR-129-5p significantly inhibited the expression of G3BP1 gene mRNA and protein level, directly targeting the 3'UTR region of G3BP1 gene. Overexpression of miR-129-5p significantly increased the protein level and phosphorylation level of p38. The expression of miR-129-5p was mediated by targeting G3BP1 and affecting the downstream p38 signaling pathway, which inhibited the proliferation of 3T3 precursor adipocytes. After overexpression of miR-129-5p, the expression of mRNA and protein of PPAR 纬 -CEBP 伪 and aP2 did not change. Mi R-129-5p did not affect the formation and number of lipid droplets and the over-expression of miR-129-5p did not affect the differentiation of adipocytes. Rosiglitazone induced a marked increase in the expression of brown fat marker genes UCP1 and Cidea in adipocytes. Overexpression of miR-129-5p significantly reduced the expression of mRNA and protein of brown fat marker gene UCP and PGC1- 伪, while overexpression of miR-129-5p inhibited the browning process of adipocytes at molecular level. In conclusion, miR-129-5p could target G3BP1 and inhibit the proliferation of 3T3 precursor adipocytes via p38 pathway, while miR-129-5p could inhibit the browning process of adipocytes at molecular level, but had no effect on the differentiation of adipocytes. The results provide a theoretical basis for the breeding of fat deposition traits and the regulation of white fat browning by miRNA.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.2

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