MicroRNA-24通过Wnt4/β-catenin信号通路促进棕色脂肪分化及分子机制研究

批准号:
81700771
项目类别:
青年科学基金项目
资助金额:
20.0 万元
负责人:
曾艳梅
依托单位:
学科分类:
H0711.脂肪组织生理调控与功能异常
结题年份:
2020
批准年份:
2017
项目状态:
已结题
项目参与者:
张倩、王丹、郑宗基、贾懿劼、吴春艳
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中文摘要
棕色脂肪分化已成为肥胖症治疗的新靶点。我们在前期研究中发现,在BMP7诱导棕色脂肪分化的过程中,miR-24表达显著上调,而增殖基因表达被明显抑制;研究发现Wnt/β-catenin信号激活可能通过促进增殖基因表达而影响脂肪分化;新近报道miR-24通过抑制Wnt4/β-catenin减轻糖尿病大鼠血管内膜增生。因此我们提出miR-24可能通过抑制Wnt4/β-catenin信号通路,从而促进棕色脂肪分化。本课题拟将棕色脂肪前体细胞miR-24过表达和沉默后探讨miR-24对棕色脂肪分化及Wnt4/β-catenin信号通路的影响;进而采用RNA-ChIP和荧光素酶报告基因等手段验证miR-24与Wnt4的直接作用关系;最后观察高脂喂养的miR-24基因敲除C57BL6小鼠的体重和糖脂代谢情况,进一步证实miR-24通过Wnt4/β-catenin影响棕色脂肪分化,进而影响肥胖及糖脂代谢。
英文摘要
Obesity, associated with various diseases, is a chronic metabolic disorder. Brown adipocyte differentiation plays a protective role in obesity. We found that the expression of miR-24 increased significantly in the process of brown adipocyte differentiation induced by BMP7, while the transcription activity of proliferational genes were inhibited in our previous study. Wnt/β-catenin signaling pathway has been reported involving in the process of adipogenesis by down-regulating the activation of proliferational genes. While recently it has been testified that miR-24 can attenuates neointimal hyperplasia in diabetic rat by inhibiting Wnt4/β-catenin signaling pathway. Thus, we hypothesize that miR-24 may promote brown adipocyte differentiation by inhibiting Wnt4/β-catenin signaling pathway. To test our hypothesis, firstly the role of miR-24 in brown adipogenesis and its effect on Wnt4/β-catenin signaling pathway will be elucidated through transfecting miR-24-mimic and miR-24-inhibitor into brown preadipocytes. Secondly, the interaction between Wnt4 and miR-24 will be analyzed by RNA-ChIP and Luciferase reporter genes. Finally, we conducted the miR-24 knockout mice with C57BL6 background ,recording the body weight and metabolism after intervention with high fat diet, to verify miR-24 can promote brown adipocyte differentiation and prevent obesity by regulating Wnt4/β-catenin signaling pathway.
前期研究发现棕色脂肪分化中mir24表达显著增加且呈时间依赖性;并发现脂肪前体细胞中细胞增殖期的mir24表达增加更显著,而增殖基因表达被明显抑制机制不明确。我们提出mir24可能通过抑制Wnt4通路调节棕色脂肪分化的假设。.为验证mir24对Wnt4的调控作用,构建出luciferase-Wnt4的荧光素酶载体,将载体质粒与mimics和 inhibitor共转染至棕色脂肪前体细胞,发现mir24mimics对Wnt4野生型载体有明显抑制作用,而mir24inhibitor对Wnt4野生型载体有明显促进作用,证实了mir-24靶向抑制靶基因Wnt4的mRNA表达。.我们将pCDH-Wnt4共转染至棕色脂肪前体细胞,使Wnt4基因过表达,采用Wnt4-siRNA的方法将细胞Wnt4基因沉默。测定vec对照组、Wnt4过表达组、shRNA对照组和Wnt4表达抑制组的增殖活性,发现Wnt4过表达后的细胞增殖活力有下降趋势;Wnt4基因沉默后细胞增殖活力有上升趋势。油红O 染色结果显示Wnt4过表达促进脂肪细胞分化,而Wnt4表达抑制则抑制脂肪细胞分化,可能与抑制细胞增殖相关。.RT-PCR结果发现Wnt4过表达组与vec对照组相比PPARr、C/EBPβ、aP2均表达增加, PGC-1a表达增加,虽UCP-1和PRDM16表达有增加趋势,但无统计学差异;而Wnt4表达抑制组与对照组相比,PPARr(P值=0.121)、C/EBPβ(P值=0.35)及PGC-1a(P值=0.4) 、UCP-1(P值=0.19)、PRDM16(P值=0.28)的mRNA表达均有下降的趋势,但无统计学差异。.蛋白水平层面结果显示Wnt4过表达组与对照组相比PPARr、C/EBPβ、aP2、PGC-1a、UCP-1和PRDM16的蛋白表达水平显著增加,具有显著统计学差异(P值为0.000,0.003,0.012,0.002, 0.002, 0.000);而Wnt4表达抑制组与对照组相比,脂肪细胞分化相关的转录因子PPARr、C/EBPβ、aP2、PGC-1a、UCP-1和PRDM16的蛋白表达水平均显著下降,有显著统计学差异(P值均<0.001)。 .我们的研究结果表明,Wnt4通路可促进棕色脂肪细胞分化并抑制细胞增殖,而miR-24通过靶向抑制Wnt4基因对棕色脂肪细胞分化起着负性调节作用。
期刊论文列表
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科研奖励列表
会议论文列表
专利列表
Genetic Analysis of SLC12A3 Gene in Chinese Patients with Gitelman Syndrome
中国Gitelman综合征患者SLC12A3基因的遗传分析
DOI:10.12659/msm.916069
发表时间:2019-08-09
期刊:MEDICAL SCIENCE MONITOR
影响因子:3.1
作者:Zeng, Yanmei;Li, Ping;Guan, Meiping
通讯作者:Guan, Meiping
DOI:10.1186/s12944-018-0896-0
发表时间:2018-11-06
期刊:Lipids in health and disease
影响因子:4.5
作者:Zeng Y;Guan M;Li C;Xu L;Zheng Z;Li J;Xue Y
通讯作者:Xue Y
国内基金
海外基金
