miR-196a-5p对3T3-L1前脂肪细胞增殖和分化的影响效应 <sup>*</sup>

doi:10.13523/j.cb.20181102

中国生物工程杂志

2018, Vol. 38

Issue (11): 9-17 DOI: 10.13523/j.cb.20181102

研究报告

miR-196a-5p对3T3-L1前脂肪细胞增殖和分化的影响效应 ^*

杨琼¹,王灵慧²,辜浩²,堵晶晶²,刘进远³,张顺华²,朱砺^2,^***(

)

^1. 成都农业科技职业学院成都 611130
^2. 四川农业大学动物科技学院成都 611130
^3. 四川省畜牧科学研究院成都 610066

The Effect of miR-196a-5p on Proliferation and Differentiation of 3T3-L1 Preadipocyte

Qiong YANG¹,Ling-hui WANG²,Hao GU²,Jing-jing DU²,Jin-yuan LIU³,Shun-hua ZHANG²,Li ZHU^2,^***(

)

^1. Chengdu Agricultural College, Chengdu 611130, China
^2. College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
^3. Sichuan Animal Science Academy, Chengdu 610066,China

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摘要：

目的:探究miR-196a-5p对小鼠前体脂肪细胞增殖、分化的影响及其潜在的分子机制。方法:(1)构建小鼠肥胖模型,RT-PCR检测脂肪组织中miR-196a-5p表达量;(2)鸡尾酒法诱导3T3-L1前脂肪细胞分化,RT-PCR检测分化过程中miR-196a-5p的表达变化;(3)合成miR-196a-5p mimics和inhibitors转染3T3-L1细胞,以CCK8、EdU试剂盒检测miR-196a-5p对3T3-L1前脂肪细胞增殖的影响作用;(4)运用油红O染色、甘油三酯测定评估miR-196a-5p对3T3-L1细胞分化的影响;(5)RT-PCR检测miR-196a-5p对前脂肪细胞增殖、分化相关基因的影响;(6)结合前人文献,运用生物信息软件、萤光素酶报告系统对miR-196a-5p调控脂肪细胞分化的靶基因进行筛选和验证。结果:(1)miR-196a-5p在肥胖小鼠脂肪组织中高表达,在3T3-L1前脂肪细胞分化过程中先升高后下降;(2)与阴性对照组相比,mimics转染抑制了3T3-L1细胞增殖,inhibitors转染促进了3T3-L1细胞增殖;(3)与阴性对照组相比,mimics组积累了大量油红着色的脂滴,甘油三酯含量增多,而inhibitors组的脂滴少而小,甘油三酯含量相对降低;(4)与阴性对照组相比,mimics转染抑制了增殖标志基因Cyclin D1、Cyclin E、CDK2和CDK4表达,促进了分化标志基因PPARγ、C/EBPα、LPL、aP2等的表达,inhibitors转染则表现出与mimics转染相反的作用;(5)miR-196a-5p可显著抑制野生型MAP4K3和MAPK1 3'UTR萤光素酶活性,而突变绑定位点可废除该抑制效应。结论:miR-196a-5p不仅可抑制3T3-L1前脂肪细胞增殖,还可促进其诱导分化、沉积脂滴;miR-196a-5p可能通过靶向调节MAP4K3和MAPK1来介导3T3-L1前脂肪细胞分化。

关键词： miR-196a-5p; 增殖; 分化; MAP4K3; MAPK1

Abstract:

Objective:To investigate the effect of miR-196a-5p on proliferation and differentiation of mouse adipocyte, and explore its potential molecular mechanisms.Methods:① Utilizing RT-PCR, miR-196a-5p expression levels in adipose tissues from obese or normal mice were measured; ② The miR-196a-5p expression level during preadipocyte differentiation were measured by RT-PCR method, after 3T3-L1 cells were induced to differentiate by cocktail method; ③After miR-196a-5p mimics or inhibitors were transfected into 3T3-L1 cells, CCK8 and EdU detection were performed to evaluate the effect of miR-196a-5p on its proliferation; ④ Measuring the effect of miR-196a-5p on 3T3-L1 cells differentiation by Oil red O staining and triglyceride assay; ⑤ Detecting the effect of miR-196a-5p on the expression levels of 3T3-L1 cells proliferation and differentiation related genes; ⑥ Based on previous reports, using bioinformatics and luciferase reporter assays to identify targets that miR-196a-5p regulates preadipocyte differentiation.Result:①miR-196a-5p not only were highly expressed in adipose tissues of obese mice, but also were expressed dynamically during 3T3-L1 cells differentiation; ②When compared with negative control, mimics transfection inhibited 3T3-L1 cells proliferation, inhibitors transfection promoted its proliferation;③When compared with negative control, mimics or inhibitors transfection increased or decreased lipid accumulation and triglyceride content, respectively; ④When compared with negative control, mimics transfection repressed proliferation related markers (Cyclin D1,Cyclin E,CDK2 and CDK4) and promoted differentiation related markers (PPARγ,C/EBPα,LPL and aP2), however, inhibitors transfection had an opposite effect than that of mimics transfection; ⑤ The miR-196a-5p mimics significantly suppressed a luciferase reporter gene whose expression was regulated by the mouse MAP4K3 and MAPK1 mRNA 3'UTR, whereas mutation of the miR-196a-5p binding site in murine MAP4K3 and MAPK1 3'UTR completely abolished this response.Conclusions:miR-196a-5p might inhibit 3T3-L1 preadipocyte proliferation, and enhance its differentiation. The regulation of preadipocyte differentiation may be mediated by targeting MAP4K3 and MAPK1.

Key words: miR-196a-5p Proliferation Differentiation MAP4K3 MAPK1

收稿日期: 2018-06-01 出版日期: 2018-12-06

ZTFLH:

Q591.5

基金资助: * 四川省科技支撑计划资助项目(2016NZ0089);四川省科技支撑计划资助项目(16ZC2838);四川省科技支撑计划资助项目(2018KZ0057)

通讯作者: 朱砺 E-mail: zhuli7508@163.com

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引用本文:

杨琼,王灵慧,辜浩,堵晶晶,刘进远,张顺华,朱砺. miR-196a-5p对3T3-L1前脂肪细胞增殖和分化的影响效应 ^*[J]. 中国生物工程杂志, 2018, 38(11): 9-17.

Qiong YANG,Ling-hui WANG,Hao GU,Jing-jing DU,Jin-yuan LIU,Shun-hua ZHANG,Li ZHU. The Effect of miR-196a-5p on Proliferation and Differentiation of 3T3-L1 Preadipocyte. China Biotechnology, 2018, 38(11): 9-17.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181102 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I11/9

表1 RT-PCR引物序列

图2 miR-196a-5p在3T3-L1前脂肪细胞分化过程中的表达

图5 MAP4K3和MAPK1是miR-196a-5p的靶基因

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