FEBS Letters 588 (2014) 429–435

journal homepage: www.FEBSLetters .org

MicroRNA-344 inhibits 3T3-L1 cell differentiation via targeting GSK3bof Wnt/b-catenin signaling pathway

0014-5793/$36.00 Crown Copyright � 2013 Published by Elsevier B.V. on behalf of Federation of European Biochemical Society. All rights reserved.http://dx.doi.org/10.1016/j.febslet.2013.12.002

⇑ Corresponding author. Fax: +86 020 39332940.E-mail address: modelin@mail.sysu.edu.cn (D. Mo).

1 These authors contributed equally to the paper.

Hu Chen 1, Siqi Wang 1, Luxi Chen, Yaosheng Chen, Ming Wu, Yun Zhang, Kaifan Yu, Zheng Huang,Lijun Qin, Delin Mo ⇑State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, PR China

a r t i c l e i n f o

Article history:Received 11 October 2013Revised 3 December 2013Accepted 3 December 2013Available online 10 December 2013

Edited by Laszlo Nagy

Keywords:miR-344GSK3bAdipogenesisWnt/b-catenin signaling

a b s t r a c t

Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of complexevents in which microRNAs might play an essential role. In this study, we found that the overexpres-sion of microRNA-344 (miR-344) inhibits 3T3-L1 cell differentiation and decreases triglyceride accu-mulation after MDI stimulation. We demonstrated that miR-344 directly targets the 30 UTR of GSK3b(Glycogen synthase kinase 3 beta). Knockdown of GSK3b with siRNA results in inhibiting 3T3-L1 dif-ferentiation, while its overexpression restores the effect of miR-344. In addition, miR-344 elevatesthe level of active b-catenin, which is the downstream effector of GSK3b in the Wnt/b-catenin signal-ing pathway. These data indicate that miR-344 inhibits adipocyte differentiation via targeting GSK3band subsequently activating the Wnt/b-catenin signaling pathway.Crown Copyright � 2013 Published by Elsevier B.V. on behalf of Federation of European Biochemical

Society. All rights reserved.

1. Introduction

Obesity is a prevalent health issue in developed countries and isusually associated with plenty of pathological disorders, includingdiabetes, hyperglycemia, hyperlipidemia, atherosclerosis and chronicinflammation [1,2]. In terms with this wide range of health hazard,the necessary to develop new and effective strategies in controllingobesity has become more acute. Obesity is the result of increase inthe adipocyte numbers and in the adipocyte size. Mature adipocytesarise from preadipocytes and progenitor cells, which reside in adi-pose tissue. Preadipocytes and progenitors undergo a terminal differ-entiation process into mature adipocytes. Therefore, controlling thisprocess could ameliorate obesity and obesity related syndrome.

MicroRNAs (miRNAs) are endogenous noncoding RNAscomprising 18–25 base pairs. miRNAs can modify gene expressionat the post-transcriptional level by complementing to the 30 untrans-lated region (UTR) of its target gene, and mediating mRNA degrada-tion or translation inhibition [3]. miRNAs have been confirmed tobe played crucial roles in diverse biological processes, including pro-liferation, differentiation, development, apoptosis, immunity, andoncogenesis [4–7]. miRNAs have also been shown to participate inadipocyte differentiation in mice, pigs, and humans [8–11].

Previously, the high throughput microarray assays found thatmiR-344 was remarkably up-regulated in 3T3-L1 cells after LiCltreatment [12]. In line with the results of microarray assays,miR-344 was up-regulated in adipogenesis after LiCl stimulation.However, our research found that miR-344 down-regulated signif-icantly during adipogenesis under normal culture conditions, sug-gesting that miR-344 plays a role in adipocyte differentiation. It ispossible that miR-344 might affect adipocyte differentiation. As weknown, Wnt/b-catenin signaling pathway can be activated by LiCland subsequently inhibit adipogenesis [13,14]. Therefore, wehypothesis that miR-344 might regulate adipogenesis throughactivating Wnt/b-catenin signaling pathway.

In this study, we demonstrated that overexpressing miR-344 couldinhibit 3T3-L1 preadipocyte differentiation into adipocyte. GSK3b, thekey component of Wnt/b-catenin signaling, was confirmed to be thetarget of miR-344, and knockdown of endogenous GSK3b by siRNAcould inhibit 3T3-L1 preadipocyte differentiation. In addition, miR-344 could increase active b-catenin, which is the downstream effec-tor of GSK3b and able to activate the transcription of genes that re-pressed preadipocyte differentiation. These results revealed amechanism of miR-344 in regulating 3T3-L1 cells differentiation.

2. Materials and methods

2.1. Cell culture and transfection

Mouse 3T3-L1 cell line and human embryonic kidney (HEK)293T cell line were purchased from the American Type Culture

Tabl

e1

Sequ

ence

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prim

ers.

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prim

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stra

nd/

prim

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quen

ce(50 -

30)

Prim

ers

for

qPCR

miR

-344

-ste

m-l

oop-

RT

CTC

AA

CTG

GTG

TCG

TGG

AG

TCG

GC

AA

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AG

AC

AG

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GU

6-st

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oop

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AA

CTG

GTG

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TGG

AG

TCG

GC

AA

TTC

AG

TTG

AG

AA

AA

ATA

TGG

miR

-344

AC

AC

TCC

AG

CTG

GG

TGA

TCTA

GC

CA

AA

GC

CT

CTC

AA

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U6

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GG

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GT

PPA

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for

gene

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ing

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C

430 H. Chen et al. / FEBS Letters 588 (2014) 429–435

Collection (ATCC). The cell lines were maintained in Dulbeccomodified Eagle medium (DMEM) supplemented with 10% fetalbovine serum (FBS), and antibiotics (100 U/mL penicillin and100 lg/L streptomycin) at 37 �C in the 5% CO2, humidified atmo-sphere. 3T3-L1 and HEK 293T cells were transfected with plasmidDNA, miRNA mimics (RIBOBIO, Guangzhou, China) and siRNA(RIBOBIO) by using Lipofectamine™ 2000 (Invitrogen, Shanghai,China) according to the manufacturer’s instruction.

2.2. 3T3-L1 induction differentiation

Adipogenic differentiation was carried out according to previ-ously published protocol. 3T3-L1 preadipocytes were grown toconfluence, 2 days later, 3T3-L1 were stimulated for 3 days in dif-ferentiation medium: DMEM containing 10% FBS and MDI(10 lg/mL insulin, 1 lM dexamethasone, and 0.5 mM IBMX). Cellswere then maintained in DMEM containing 10% FBS and 10 lg/mLinsulin. The medium was replaced every other day. Mature adipo-cytes were visualized by staining with Oil Red O solution.

2.3. Oil red O staining

Cells were washed with PBS and fixed with 4% paraformaldehydefor 10 min. For Oil Red O quantitative analysis, the intracellular ab-sorbed Oil Red O was extracted in 100% isopropanol, and absorbancewas measured at 510 nm wave length.

2.4. Plasmid construction

For GSK3b 30 UTR reporter assay, the potential binding sites se-quence were obtained by annealing sense and antisense strand(Table 1), and then the annealed sequences were inserted down-stream of the stop codon of Renilla luciferase gene in psiCHECK-2dual-luciferase reporter plasmid (Promega, Madison, USA), desig-nated as psiCHE-GSK3b 30 UTR-wt. psiCHE-GSK3b 30 UTR-mut,which carried a mutated sequence in the putative binding sitefor miR-344, was generated by randomly disorganized seed regionof annealed sequences.

To construct GSK3b overexpression vector (3.1-GSK3b), GSK3bcoding sequence was amplified by PCR (primers in Table 1) andthen cloned into NheI and XhoI site in pcDNA3.1 vector(Invitrogen).

2.5. Luciferase reporter assay

HEK 293T cells were plated in 48-well plates at a density of6 � 104 cells per well. Cells were transfected with 50 ngpsiCHECK-2 recombination vector and miR-344 mimics or miRNAmimics Negative Control. Four replicates were made for eachtransfection. Firefly and Renilla luciferase activities were measuredwith the Dual-Glo luciferase system (Promega) and measured withSynergyTM 2 multi-mode readers (BioTek, Vermont, USA) at48 hours after transfection.

2.6. RNA isolation and qPCR

Total RNA was extracted using TRIzol reagent (Invitrogen)according to the manufacturer’s instructions. First-strand cDNAwas obtained using the Reverse Transcription System (Promega),Oligo(dT) and Stem-loop Reverse Transcription primer for mRNAand miRNA, respectively. Quantitative real-time PCR (qPCR) wasperformed using SYBR Premier Dimer Eraser™ (TaKaRa, Dalian, Chi-na) on a LightCycler 480 (Roche, Basel, Switzerland), and relativequantification (2�44Ct) method was used to analyze the data.Endogenous NoNo mRNA was used as reference for mRNA quantifi-cation, as it was expressed stable in murine [15], and U6 for miRNA.

H. Chen et al. / FEBS Letters 588 (2014) 429–435 431

2.7. Western blot

Cells were lysed in cell lysis buffer (Beyotime, Shanghai, China) inthe presence of a protease inhibitor cocktail (Sigma, Shanghai, Chi-na) and phosphatase inhibitors (5 mM Na4P2O7, 50 mM NaF, 1 mMvanadate), and protein content was quantified by BCA methods[16]. 40 lg total cellular protein was fractionated by 12% (w/v)SDS–PAGE and electronically transferred to 0.45 lm PVDFmembrane (Roche). The membrane was blocked for 2 hours inTBS-Tween20 (TBST) containing 5% (w/v) skimmed milk, andincubated with primary antibody for 1 hour at room temperature.The membrane was washed with TBST and incubated for 1 hourwith secondary antibody. Blots were visualized using a commercialenhanced chemiluminescene (ECL) detection Kit (Thermo Scientific,Guangzhou, China). GSK3b was measured by monoclonal anti-GSK3b antibody (#9315, CST, Shanghai, China). Active b-catenin(non-phosphorylation) was detected by monoclonal anti-Non-phospho b-catenin antibody (Ser33/37/Thr41) (#8814, CST).Monoclonal anti-b-catenin antibody (#8480, CST) was used to de-tect total b-catenin protein. Cyclin D1 was detected by monoclonalCyclin D1 antibody (#2926, CST). GAPDH protein was used asreference (sc-59540, Santa Cruz Biotechnology, Shanghai, China),and the secondary antibody was HRP-conjugated goat anti-rabbitIgG (#7074s, CST) and HRP-conjugated goat anti-mouse IgG(#7076s, CST).

2.8. Statistical analysis

All results are presented as mean ± standard errors of the means(SEM) based on at least three separate experiments. Unless other-wise noted, the differences between groups were analyzed byusing a Student’s two-tailed t test when only two groups werecompared or by ANOVA when more than two groups were com-pared. Differences were regarded as significant at a P value of<0.05.

Fig. 1. Up-regulation of miR-344 in LiCl stimulated 3T3-L1 cells. (A) 3T3-L1 preadipocytewas initiated after 48 hours treatment. Cells were fixed and stained with Oil Red O on daymeasured the absorbance at 510 nm wave length. (C) miR-344 was detected during 3treatment by using Stem-loop qPCR. Data are means of at least three independent expe

3. Results

3.1. miR-344 was significantly up-regulated during 3T3-L1 cellsdifferentiation after LiCl treatment

The preadipocytes which treated with LiCl failed to differentiateinto mature adipocytes after MDI stimulation, as confirmed by OilRed O staining and quantitative analysis (Fig. 1A and B). As acontrol, NaCl was used in place of LiCl before MDI stimulation,the preadipocytes differentiated normally into adipocytes. Todetermine the changes of miR-344 during 3T3-L1 cells differentia-tion, cells were collected at different time points, including �1 day(�1D), 0D (MDI stimulation), 6 hour (H), 12H, 1D, 2D, 4D, and 6D.Stem-loop qPCR results showed that miR-344 was significantlydown-regulated after MDI stimulation on 0D under normal cultureconditions, and maintained low level in adipogenesis (Fig. 1C).However, Compared with NaCl group, miR-344 was markedlyup-regulated on �1D, 12H, 1D and 2D after LiCl treatment(Fig. 1D). In addition, the expression of miR-344 also increasedon 0D, 6H, 4D, 6D, although these differences did not reach statis-tical significance (Fig. 1D). These results indicated that miR-344might inhibit 3T3-L1 preadipocyte differentiation.

3.2. miR-344 repressed 3T3-L1 preadipocytes differentiation

To investigate the role of miR-344 in adipogenesis, 3T3-L1 pre-adipocytes were transfected with miR-344 mimics or miRNA mim-ics Negative Control (NC) before adipogenic stimulation. Usingstem-loop qPCR assay, it was found that a 400-fold increase formature miR-344 in the transfected preadipocytes (P < 0.01)(Fig. 2C). As shown in Fig. 2A, miR-344 strongly inhibited 3T3-L1preadipocytes differentiating into mature adipocytes, as demon-strated by a reduction of the number of Oil Red O positive cells.In contrast, the irrelevant Negative Control did not affectadipogenic differentiation. Quantitative analysis of intracellular

s were grown to confluence and treated with LiCl or NaCl, adipogenic differentiation6 after MDI stimulation; (B) quantified lip accumulation by extracting Oil Red O andT3-L1 cells differentiation under normal culture conditions, and (D) LiCl or NaClriments; error bars indicate SEM. ⁄P < 0.05; ⁄⁄P < 0.01.

432 H. Chen et al. / FEBS Letters 588 (2014) 429–435

accumulation neutral lipids revealed statistically significantinhibition of adipocyte formation (P < 0.01) (Fig. 2B). Severalwell-defined key transcription factors of adipogenic differentiationwere also detected during differentiation by qPCR. Compared withNegative Control group, the expression of PPARc, C/EBPa, FABP4and Adiponectin genes were significantly down-regulated in cellstransfected with miR-344 mimics (Fig. 2D–G). Collectively, these

Fig. 2. miR-344 inhibited 3T3-L1 preadipocytes differentiation. (A) 3T3-L1 preadipocy(50 nM) and NC (50 nM), adipogenic differentiation was initiated at 24 hours post-transfequantified lip accumulation by extracting Oil Red O and measured the absorbance at 510were induced to undergo differentiation. After 24 hours post-transfection, miR-344 transfCEBPa, FABP4 and Adiponectin at �1D, 0D, 2D, 4D, 8D, and 10D after MDI stimulation. D⁄P < 0.05; ⁄⁄P < 0.01.

Fig. 3. GSK3b was a direct target of miR-344. (A) The predicted binding site of miR-344reporter vector psi-CHECK2 at the 30 end of hRluc (Renilla luciferase gene), the constipsiCHECK2, psiCHE-GSK3b 30 UTR wt or psiCHE-GSK3b 30 UTR mut vector was co-transfecwas determined. (C) GSK3b mRNA measured by qPCR, and (D) protein expression dpreadipocytes at 72 hours post-transfection with miR-344 mimics. Data are means of at

results showed that miR-344 could inhibit 3T3-L1 preadipocytedifferentiation.

3.3. miR-344 directly targeted 30 UTR of GSK3b

In order to reveal the mechanisms by which miR-344 sup-presses 3T3-L1 preadipocyte differentiation, we focused initially

tes were grown to confluence and transiently transfected with miR-344 mimicsction. Cells were fixed and stained with Oil Red O on day 6 after MDI stimulation; (B)nm wave length. (C) 3T3-L1 preadipocytes transfected with miR-344 mimics or NC

ection efficiency was determined by stem-loop qPCR. (D) And qPCR analyzed PPARc,ata are means of at least three independent experiments; error bars indicate SEM.

in the 30 UTR of GSK3b (up). The GSK3b 30 UTR was inserted into the dual-luciferasetutive firefly luciferase (hluc+) was used as an internal control (bottom). (B) Theted with miR-344 or NC into HEK293 cells, and normalized Renilla luciferase activityetected by western blotting (up) and densitometry analysis (bottom) in 3T3-L1least three independent experiments; error bars indicate SEM. ⁄P < 0.05; ⁄⁄P < 0.01.

H. Chen et al. / FEBS Letters 588 (2014) 429–435 433

on identifying the potential targets of miR-344. The genes involvedin Wnt/b-catenin signaling pathway got more attention, becausemiR-344 was up-regulated after LiCl treatment, furthermore, LiClis able to activate Wnt/b-catenin signaling pathway which inhibitsadipogenesis. Consequently, GSK3b, a key component of Wnt sig-naling pathway, was predicted by TargetScan (http://www.target-scan.org), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybird/), and miRanda (http://www.microrna.org/microrna/getGeneForm.do) analysis as a prime target (Fig. 3A, up).

To confirm mouse GSK3b as a target of miR-344 actually, wesynthetized sequences of wild-type 30 UTR of mouse GSK3b con-taining putative binding site for miR-344 and the mutant 30 UTRwith a 7 bp mutation in the seed region. After anneal, the se-quences were inserted downstream of the luciferase gene(Fig. 3A, bottom) and luciferase assays were performed in HEK293T cells. As shown in Fig. 3B, compared with Negative Controlgroup, co-transfection of miR-344 mimics with the mouse GSK3b30 UTR wild-type reporter (psiCHE-GSK3b 30 UTR-wt) resulted ina highly significant decrease in luciferase activity (P < 0.05). Con-sistent with the result, no decrease in luciferase activity was ob-served when miR-344 mimics or Negative Control co-transfectedwith the empty vector or the mutant reporter (psiCHE-GSK3b 30

UTR-mut), indicating that the predicted site is a direct target ofmiR-344.

Fig. 4. Overexpression GSK3b could restore the effect of miR-344. (A) Transfection of tGSK3b mRNA (left) and protein (right), detected by qPCR and western blotting, (B) whquantitation). (C) Overexpression of GSK3b through transfection of GSK3b expression velevels was detected. (D) 3T3-L1 preadipocytes were grown to confluence, co-transfectedpcDNA3.1; adipogenic differentiation was initiated at 24 hours post-transfection. Cellquantitative analysis Oil Red O (right). (E) qPCR analysis mRNA expression of PPARc, Cpost-transfection with miR-344 mimics into 3T3-L1 preadipocytes, qPCR analysis b-cateb-catenin) and Cyclin D1 protein levels (middle), and the differences were analyzed byerror bars indicate SEM. ⁄P < 0.05; ⁄⁄P < 0.01.

To examine whether the miR-344 can regulate the expression ofGSK3b or not, GSK3b mRNA and protein level were measured in3T3-L1 cells transiently transfected with miR-344 mimics by qPCRand western blotting, respectively. As a result, miR-344 inhibitedendogenous GSK3b protein expression in 3T3-L1 preadipocytes(Fig. 3D), even though no significant inhibition was detected atthe mRNA level (Fig. 3C). Taken together, these results indicate thatGSK3b is a direct and authentic target of miR-344.

3.4. miR-344 suppressed adipogenesis through targeting GSK3b andactivating Wnt/b-catenin signaling pathway

GSK3b was shown to be an authentic target of miR-344, but fur-ther investigation was needed to determine whether miR-344inhibited 3T3-L1 differentiation through direct down-regulationGSK3b. Firstly, we investigated whether reducing GSK3b expres-sion might resemble the repression effect of miR-344 overexpres-sion. After transfection of siGSK3b into 3T3-L1 preadipocytes, itwas not only substantially knocked down the expression of GSK3bmRNA and protein (Fig. 4A), but also resulted in significantlysuppression of 3T3-L1 preadipocytes differentiation (Fig. 4B). Thisresult is in line with the influence of GSK3b inhibitors, which cansuppress GSK3b protein expression and impair the adipocyte dif-ferentiation [17–19]. Thus, disruption of GSK3b inhibited 3T3-L1

hree different siGSK3b into 3T3-L1 preadipocytes to knockdown the expression ofich resulted in suppressed differentiation of 3T3-L1 cells (Oil Red O staining andctor (3.1-GSK3b) into 3T3-L1 preadipocytes, GSK3b mRNA (left) and protein (right)with miR-344 and 3.1-GSK3b, NC and pcDNA3.1, NC and 3.1-GSK3b, miR-344 and

s were fixed and stained with Oil Red O on day 6 after MDI stimulation, thenEBPa, FABP4 and Adiponectin from corresponding transfections. (F) After 72 hoursnin mRNA level (left), western blotting detected b-catenin, active b-catenin (Non-Pdensitometry analysis. Data are means of at least three independent experiments;

TCF

Pro-adipogenic

Groucho

Dvl

β-cateP P

CK1

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APC

Axinβ-cate

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LRP FzdDkkA

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Anti-adipogenic

Groucho

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Anti-adipogenic

Groucho

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CK1

Gsk3β

APC

Axinβ-cate

P P

miR-344

CK1

APC

Axinβ-cate

Gsk3β ORF5’ 3’

LRP FzdC

Fig. 5. Schematic illustration of miR-344 regulates 3T3-L1 cells differentiation. (A) In the absence of a Wnt signal, Dkk1 inhibits Wnt signaling by binding as a high-affinityantagonist to LRP co-receptors. b-Catenin is captured by a destruction complex that contains Axin and APC (adenomatous polyposis coli), which facilitates b-cateninphosphorylation by CK1 (casein kinase I) and GSK3b. Then phosphorylated b-catenin was recognized by E3 ubiquitin ligase, this cause b-catenin to be degraded by theproteasome. (B) Interaction of a Wnt ligand (Wnt10b) with its specific receptor complex containing a frizzled (Fzd) family and LRP5/6 co-receptors triggers the formation ofDvl-Fzd complexes and the phosphorylation of LRP, facilitating relocation of Axin to the membrane and inactivation of the destruction box [28]. This results inhypophosphorylation of b-catenin, which accumulates in the cytoplasm and translocates to the nucleus, where it binds to TCF/LEF transcription factors to activate b-catenin-dependent genes and inhibit adipogenesis by suppressing the transcription of adipogenic genes. (C) miR-344 can target GSK3b mRNA and repression it translation, whichresults in b-catenin hypophosphorylation and accumulation in cytoplasm. Subsequently, b-catenin translocates into the nucleus and activates the transcription of geneswhich suppressed adipogenic genes expression.

434 H. Chen et al. / FEBS Letters 588 (2014) 429–435

preadipocytes differentiation, similar to the result of miR-344overexpression.

Furthermore, we examined whether constitutive expression ofGSK3b could counteract the repression effect of miR-344. Overex-pression of GSK3b by transfecting 3.1-GSK3b, as evidenced byraised expression of GSK3b mRNA and protein (Fig. 4C), resultedin no apparent change in 3T3-L1 cells differentiation efficiency(Fig. 4D, 3.1-GSK3b + NC vs. pcDNA3.1 + NC). However, GSK3bwas confirmed to be a target of miR-344 during adipocyte differen-tiation, its role was further assessed by its overexpression in3T3-L1 preadipocytes in the presence of transfected miR-344,which increased 3T3-L1 cells differentiation efficiency (Fig. 4D,3.1-GSK3b+miR-344). As expected, the presence of miR-344 withcontrol empty plasmid (pcDNA3.1) in 3T3-L1 preadipocytes ledto suppression of 3T3-L1 cells differentiation (Fig. 4D). Further-more, the decrease in adipogenic marker gene expression (PPARc,CEBPa, FABP4 and Adiponectin) as a result of miR-344 transfectionwas attenuated by co-expression of GSK3b (Fig. 4E). Therefore, theoverexpression of GSK3b could restore adipocytes differentiationand overcome the effect of miR-344.

In addition, b-catenin, the substrate of GSK3b in Wnt/b-cateninsignaling, mRNA level was notably augmented (P < 0.01 ) aftertransfected with miR-344 mimics (Fig. 4F, left). Although the pro-tein level of b-catenin was increased, the difference did not reachstatistical significance (P = 0.287) (Fig. 4F middle and right). Tothe best of our knowledge, GSK3b modulated b-catenin throughphosphorylating it at Thr-41, Ser-37 and Ser-33 [20]. So wedetected the active b-catenin (non-phosphorylation) by anti-Non-phospho b-catenin (Ser33/37/Thr41). In a marked contrast, theactive b-catenin was significantly increased (P < 0.01) whilemiR-344 overexpression (Fig. 4F, middle and right). Generally,large amounts of b-catenin in cytoplasm might translocate intothe nucleus, and then stimulate b-catenin-dependent genestranscription which participated in inhibiting adipogenic genesexpression. Furthermore, we found that Cyclin D1, a well-knowntarget of b-catenin, was appreciably up-regulated (P < 0.01) whenactive b-catenin increased (Fig. 4F, middle and right). Collectively,

miR-344 inhibited GSK3b protein translation and increasedb-catenin level, and the latter might translocate into nucleus andactivate the transcription of genes that repressed adipocytesdifferentiation.

4. Discussion

In recent years, a number of miRNAs have been found as a classof modulator in gene expression regulation through altering mRNAor translation level [3]. Many studies have shown that miRNAs mayfunction as either pro-adipogenesis or anti-adipogenesis genes.miR-344 was first isolated from rat embryonic primary corticalneurons [21]. In mouse, miR-344 encoding gene located nearbythe mouse 7C imprinted domains, strongly suggesting animprinted expression [22]. However, the function of miR-344 isunclear so far. Our previous research implicated miR-344 might in-volve in regulating adipocyte differentiation [12]. In this study, wedemonstrated that miR-344 inhibit 3T3-L1 preadipocyte differenti-ation through down-regulating the expression of GSK3b by target-ing the 30 UTR of GSK3b (Fig. 3B and D).

GSK3b is a serine/threonine kinase that originally identified as aregulator of cell metabolism but participated in a number ofbioprocesses, for instance, protein synthesis, cell proliferation, celldifferentiation, microtubule dynamics and cell motility by phos-phorylating initiation factors [23]. Disruption GSK3b protein expres-sion by GSK3b inhibitors (i.e. LiCl, BIO [17], and SB415286 [18])could inhibited preadipocyte differentiation. Presently, we demon-strated that knockdown GSK3b could suppress 3T3-L1 preadipocytedifferentiation (Fig. 4B). Conversely, overexpression GSK3b cancounteract the inhibition effect of miR-344 on adipocyte differenti-ation (Fig. 4D). In fact, GSK3b is a key component of Wnt/b-cateninsignaling pathway. GSK3b destabilizes b-catenin, which is a keydownstream effector in the Wnt/b-catenin signaling, by phosphory-lating it at Thr-41, Ser-37 and Ser-33. Then phosphorylatedb-catenin can be recognized by E3 ubiquitin ligase, this causeb-catenin to be degraded by the proteasome thus the transcriptionaltargets of b-catenin remain off. Conversely, decreasing GSK3b could

H. Chen et al. / FEBS Letters 588 (2014) 429–435 435

facilitate the accumulation of b-catenin in cytoplasm [14]. Weshowed here that miR-344 decreased GSK3b protein expression(Fig. 3D), and elevated active b-catenin (Non-P b-catenin) (Fig. 4F).This allows b-catenin to accumulate in cytoplasm and enter the nu-cleus, where it stimulates the transcription of b-catenin-dependentgenes that result in decrease of adipogenic genes expression. As weknow, Cyclin D1 was a well-known b-catenin target gene [24]. Farm-er group found that augmented b-catenin level increased Cyclin D1[25], which can inhibit PPARc-mediated adipogenesis [26,27]. Theregulatory mechanism of miR-344 can be illuminated in the model(Fig. 5).

In conclusion, our study has proved the fundamental role andmechanism of miR-344 in the suppression of adipocyte differenti-ation, at least in part, by inhibiting GSK3b at the post-transcrip-tional level and activating the transcription of the Wnt/b-cateninsignaling pathway downstream genes that decrease the expressionof adipogenic genes. Therefore, miR-344 and its target genes maypotentially play a role in the pathological progression of obesity-related diseases.

Acknowledgements

This research was supported by the Joint Funds of NSFC-Guang-dong (U1201213), the National Natural Science Foundation ofChina (31272417) and China Agriculture Research System(CASR-36).

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