本文選題：miR-129-5p + 脂肪細(xì)胞�。� 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：MicroRNA在脂肪的各個(gè)進(jìn)程中都發(fā)揮著重要作用,包括脂肪細(xì)胞的增殖、分化以及棕色化。MiR-129-5p是一個(gè)重要的腫瘤抑制因子,它對(duì)很多癌細(xì)胞的增殖具有抑制作用。中南大學(xué)關(guān)于mi RNA的微陣列分析表明miR-129-5p在胰島素抵抗的3T3細(xì)胞中高表達(dá),預(yù)示著mi R-129-5p可能在脂肪細(xì)胞中發(fā)揮作用。本文旨在探索miR-129-5p在脂肪細(xì)胞增殖、分化及棕色化過(guò)程中發(fā)揮的作用。本文使用miR-129-5p mimic轉(zhuǎn)染3T3細(xì)胞系和小鼠原代皮下脂肪細(xì)胞,并使用EdU、流式細(xì)胞術(shù)、CCK-8、油紅O染色、Bodipy染色、RT-qPCR、Western blot等技術(shù)檢測(cè)過(guò)表達(dá)miR-129-5p對(duì)脂肪細(xì)胞增殖、分化及棕色化的影響。同時(shí)構(gòu)建了miR-129-5p靶基因G3BP1的熒光素酶報(bào)告載體,對(duì)miR-129-5p調(diào)控3T3增殖的機(jī)制進(jìn)行探索。獲得的主要結(jié)果如下:1.在人、小鼠、大鼠等物種間miR-129-5p的成熟序列及種子序列一致;miR-129-5p在小鼠腦、皮下白色脂肪、棕色脂肪中表達(dá)較高,且其表達(dá)水平在脂肪細(xì)胞增殖期間呈下降趨勢(shì),在分化期間呈升高趨勢(shì)。2.過(guò)表達(dá)miR-129-5p可以影響cyclin D及p27等細(xì)胞周期相關(guān)基因的mRNA及蛋白水平表達(dá);miR-129-5p顯著減少了處于細(xì)胞周期S期的細(xì)胞數(shù)目同時(shí)顯著增加了處于細(xì)胞周期G2期的細(xì)胞數(shù)目并造成G2期阻滯;過(guò)表達(dá)miR-129-5p在分子水平和細(xì)胞水平上顯著抑制了3T3前體脂肪細(xì)胞的增殖過(guò)程。3.過(guò)表達(dá)miR-129-5p顯著抑制了G3BP1基因mRNA及蛋白水平的表達(dá);mi R-129-5p直接靶定G3BP1基因的3'UTR區(qū)域;過(guò)表達(dá)miR-129-5p顯著升高了p38的蛋白水平及磷酸化水平表達(dá);miR-129-5p是通過(guò)靶定G3BP1并影響下游p38信號(hào)通路發(fā)揮其抑制3T3前體脂肪細(xì)胞增殖作用的。4.過(guò)表達(dá)miR-129-5p后成脂相關(guān)基因PPARγ、CEBPα及aP2的mRNA及蛋白水平的表達(dá)沒(méi)有發(fā)生變化;mi R-129-5p沒(méi)有影響脂滴的形成及數(shù)量;mi R-129-5p過(guò)表達(dá)不影響脂肪細(xì)胞分化過(guò)程。5.羅格列酮法誘導(dǎo)小鼠脂肪細(xì)胞棕色化后棕色脂肪標(biāo)志基因UCP1及Cidea的表達(dá)明顯升高。過(guò)表達(dá)miR-129-5p使棕色脂肪標(biāo)志基因UCP及PGC1-α的mRNA和蛋白水平表達(dá)顯著降低;過(guò)表達(dá)miR-129-5p在分子水平上抑制了脂肪細(xì)胞棕色化過(guò)程。綜上所述,mi R-129-5p可以通過(guò)靶定G3BP1并影響p38通路抑制3T3前體脂肪細(xì)胞的增殖過(guò)程,同時(shí)miR-129-5p可以在分子水平上抑制脂肪細(xì)胞的棕色化過(guò)程,但對(duì)脂肪細(xì)胞的分化過(guò)程沒(méi)有影響。該研究結(jié)果為脂肪沉積性狀的選育以及miRNA對(duì)白色脂肪棕色化的調(diào)控研究提供了理論依據(jù)。
[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 緯 -CEBP 偽 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- 偽, 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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 劉寒梢;馬越云;肖華勝;;血清微小RNA(miR-129-3p、miR-767-3p和miR-877*)在結(jié)直腸癌診斷中的價(jià)值[J];腫瘤;2012年01期

2 ;MAPK signal pathways in the regulation of cell proliferation in mammalian cells[J];Cell Research;2002年01期

，

本文編號(hào)：1822596