MiR-186-5p对3T3-L1前脂肪细胞增殖分化的影响研究 <sup>*</sup>

doi:10.13523/j.cb.1907035

中国生物工程杂志

2020, Vol. 40

Issue (3): 21-30 DOI: 10.13523/j.cb.1907035

研究报告

MiR-186-5p对3T3-L1前脂肪细胞增殖分化的影响研究 ^*

辜浩^1,²,郭鑫宇^1,²,堵晶晶^1,²,张锫文^1,²,王定国³,廖坤⁴,张顺华^1,²,朱砺^1,^2,^**(

)

1 四川农业大学动物科技学院成都 611130
2 四川农业大学畜禽遗传资源发掘与创新利用四川省重点实验室
3 成都市动物疫病预防控制中心成都 610041
4 四川省通江县农业局畜牧站巴中 636600

The Effect of miR-186-5p on the Proliferation and Differentiation of 3T3-L1 Preadipocyte

GU Hao^1,²,GUO Xin-yu^1,²,DU Jing-jing^1,²,ZHANG Pei-wen^1,²,WANG Ding-guo³,LIAO Kun⁴,ZHANG Shun-hua^1,²,ZHU Li^1,^2,^**(

)

1 Sichuan Agricultural University College of Animal Science and Technology, Chengdu 611130,China
2 Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province,Sichuan Agricultural University, Chengdu 611130,China
3 Chengdu animal disease prevention and control center, Chengdu 610041,China
4 Agricultural Bureau Animal Husbandry Station of Tongjiang, Bazhong 636600,China

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摘要：

目的探究miR-186-5p对小鼠3T3-L1前脂肪细胞增殖,分化的影响及其潜在的分子机制.方法: qRT-PCR检测miR-186-5p在不同周龄小鼠白色脂肪组织及3T3-L1前脂肪细胞增殖分化过程中的表达变化;通过脂质体将miR-186-5p mimics,inhibitors转染入增殖液或分化液培养的3T3-L1细胞后,利用CCK-8,EdU和qRT-PCR检测3T3-L1前脂肪细胞增殖变化,油红O染色观察其脂滴形态;通过生物信息软件TargetScan和双荧光报告系统分别对miR-186-5p靶基因进行预测和确认.结果: (1)miR-186-5p在1~6周龄小鼠的白色脂肪组织及3T3-L1前脂肪细胞自然分化过程中表达量均逐渐上调.(2)与阴性对照相比,mimics或inhibitors转染分别显著地促进或抑制了miR-186-5p的表达.(3)过表达miR-186-5p后,3T3-L1前脂肪细胞的增殖速率减慢,脂滴增大增多;而抑制miR-186-5p后,3T3-L1前脂肪细胞增殖速率增快,脂滴数量减少,且粒径变小.其中过表达miR-186-5p显著地降低了野生型Wnt5a和Mapk1 3'-UTR活性,而突变相应的绑定位点可解除该抑制作用.结论: miR-186-5p可抑制3T3-L1前脂肪细胞增殖,且通过直接靶向Wnt5a和Mapk1以促进其分化为成熟脂肪细胞.

关键词： 3T3-L1前脂肪细胞; miR-186-5p; 分化; 增殖; Wnt5a; Mapk1

Abstract:

Objective: To investigate the effect of miR-186-5p on the proliferation and differentiation of mouse 3t3-L1 preadipocytes and its potential molecular mechanism.Methods: qRT-PCR was utilized to measure the expression levels of miR-186-5p in adipose tissue of different-age mice or during the proliferation and differentiation of 3T3-L1 preadipocyte; miR-186-5p mimics or inhibitors were transfected into the 3T3-L1 adipocytes cultured in growth or differentiation medium using lipofectamine; CCK-8,EdU, qRT-PCR or Oil red O staining were performed to determine the effect of miR-186-5p on 3T3-L1 preadipocyte proliferation or differentiation, respectively. Meanwhile, TargetScan and dual-luciferase assay were used to predict and confirm the target genes of miR-186-5p.Results: With aging of mice or 3T3-L1 preadipocyts differentiation into mature adipocyte, the mRNA levels of miR-186-5p were upregulated gradually. When compared to the negative control, notably, mimics or inhibitors transfection could remarkably increase or decrease the expression levels of miR-186-5p, respectively. MiR-186-5p overexpression significantly inhibited the 3T3-L1 preadipocyte proliferation and promoted its differentiation, and in contrast, miR-186-5p inhibition had opposite effect on 3T3-L1 preadipocyte proliferation and differentiation compared to the overexpression group. Further,overexpression of miR-186-5p significantly repressed the relative luciferase activity of wild type-Wnt5a and Mapk1 3'-UTR were found, whereas its inhibition effect was abolished by the mutation of the binding sites.Conclusions: miR-186-5p inhibits the proliferation of 3T3-L1 preadipocyte and promotes its differentiation by targeting Wnt5a and Mapk1.

Key words: 3T3-L1 preadipocyte miR-186-5p Differentiation Proliferation Wnt5a Mapk1

收稿日期: 2019-07-17 出版日期: 2020-04-18

ZTFLH:

Q819

基金资助: * 国家重点研发计划项目(2018YFD0501004);四川省科技支撑项目(2016NZ0050);国家现代农业产业技术体系四川生猪创新团队项目(SCCXTD-008)

通讯作者: 朱砺 E-mail: zhuli7508@163.com

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引用本文:

辜浩,郭鑫宇,堵晶晶,张锫文,王定国,廖坤,张顺华,朱砺. MiR-186-5p对3T3-L1前脂肪细胞增殖分化的影响研究 ^*[J]. 中国生物工程杂志, 2020, 40(3): 21-30.

GU Hao,GUO Xin-yu,DU Jing-jing,ZHANG Pei-wen,WANG Ding-guo,LIAO Kun,ZHANG Shun-hua,ZHU Li. The Effect of miR-186-5p on the Proliferation and Differentiation of 3T3-L1 Preadipocyte. China Biotechnology, 2020, 40(3): 21-30.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1907035 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I3/21

表1 引物信息

图1 miR-186-5p在各组织,3T3-L1前脂肪细胞中的表达量

图2 miR-186-5p对3T3-L1前脂肪细胞增殖的影响

图3 miR-186-5p对3T3-L1前脂肪细胞分化的影响

图4 miR-186-5p同源性分析及潜在靶基因预测

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