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

doi:10.13523/j.cb.20181102

China Biotechnology

2018, Vol. 38

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

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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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

Received: 01 June 2018 Published: 06 December 2018

ZTFLH:

Q591.5

Corresponding Authors: Li ZHU E-mail: zhuli7508@163.com

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	Qiong YANG
	Ling-hui WANG
	Hao GU
	Jing-jing DU
	Jin-yuan LIU
	Shun-hua ZHANG
	Li ZHU

Cite this article:

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.

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

Table 1 The primer sequences used for qRT-PCR

Fig.1 miR-196a-5p highly expressed in adipose tissues of obese mice RT-PCR measured the expression of miR-196a-5p in adipose tissues including gonadal fat (g-Fat), inguinal fat (i-Fat) and perirenal fat (p-Fat) from HFD- and NCW-fed mice. ^* P<0.05; ^** P<0.01

Fig.2 The expression of miR-196a-5p during 3T3-L1 preadipocyte differentiation RT-PCR measured the expression levels of miR-196a-5p at day 0, 2, 4, 6, 8 of preadipocyte differentiation. ^* P<0.05; ^** P<0.01

Fig.3 miR-196a-5p inhibited 3T3-L1 preadipoyte proliferation3T3-L1 cells were transfected with miR-196a-5p mimics, inhibitors, or negative control (NC) (a) Expression levels of miR-196a-5p measured by RT-PCR. Effects of the miR-196a-5p mimics, inhibitors and control on 3T3-L1 cells proliferation were determined by (b) CCK-8 and (c,d) Edu assays. RT-PCR measured the expression levels of Cyclin D1, Cyclin E, CDK2 and CDK4. ^* P< 0.05; ^** P<0.01

Fig.4 miR-196a-5p promoted 3T3-L1 preadipocyte differentiation (a) qRT-PCR measured the expression levels of miR-196a-5p after transfection with the mimics, inhibitors or negative control (NC) (b) Transfected and differentiated cells stained with oil red O (c) Triglyceride content measured by spectrophotometric analysis (d) RT-PCR determined the expression levels of markers related adipocyte differentiation. ^* P<0.05; ^** P<0.01

Fig.5 MAP4K3 and MAPK1 were target genes of miR-196a-5p Sequence alignment of miR-204-5p with the 3' UTR of mouse (mmu) MAP4K3 (a) and MAPK1 (b) mRNA. The binding site and seed region of miR-196a-5p are indicated in red. Expression levels of MAP4K3 (c) and MAPK1 (d) in transfected and differentiated cells were measured by RT-PCR. (e),(f) A recombinant double fluorescent reporter plasmid containing the luciferase gene under the control of either the wild-type (WT) 3' UTR of MAP4K3 and MAPK1, or a mutant sequence (MUT) were used to investigate the interaction of miR-196a-5p with the 3' UTR of MAP4K3 and MAPK1 mRNA. This assay revealed the repressive effect of miR-196a-5p on MAP4K3 and MAPK1 activity. ^* P<0.05; ^** P<0.01


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