A signal-amplification circuit between miR-218 and Wnt/β-catenin signal promotes human adipose...

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Bone xxx (2013) xxx–xxx

BON-10157; No. of pages: 8; 4C:

Contents lists available at ScienceDirect

Bone

j ourna l homepage: www.e lsev ie r .com/ locate /bone

Original Full Length Article

A signal-amplification circuit between miR-218 and Wnt/β-cateninsignal promotes human adipose tissue-derived stem cellsosteogenic differentiation

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Wei-Bing Zhang a,b,⁎, Wei-Jie Zhong c, Lin Wang a,b

a Institute of Stomatology, Nanjing Medical University, Nanjing, Chinab Department of Orthodontics, School of Stomatology, Nanjing Medical University, Nanjing, Chinac Department of Orthodontics, Shanghai Stomatological Disease Center, Shanghai, China

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Abbreviations:miRNA, microRNA; UTR, untranslated rqRT-PCR, quantitative reverse transcription-polymerasemarrow-derived mesenchymal stem cells; hASCs, humOCN, osteocalcin; BSP, bone sialoprotein; ALP, alkaline phtranscription factor 2; SFRP2, secreted frizzled related proprotein 2.⁎ Corresponding author at: Institute of Stomatology

Nanjing, China.E-mail address: [email protected] (W.-B. Zh

8756-3282/$ – see front matter © 2013 Published by Elsehttp://dx.doi.org/10.1016/j.bone.2013.09.015

Please cite this article as: Zhang W-B, et al, Atissue-derived stem cells osteogenic differen

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Article history:Received 18 May 2013Revised 19 September 2013Accepted 23 September 2013Available online xxxx

Edited by: David Fyhrie

Keywords:MicroRNAHuman adipose-derived mesenchymal stemcellsOsteogenic differentiationmiR-218Wnt

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ECTED PHuman adipose-derived stem cells (hASCs) have become a highly attractive source of seed cells in bone regener-

ative. It has become a key issue how to effectively improve osteogenic differentiation of hASCs in the bone tissueengineering.Numerous regulatory pathways dominate osteogenic differentiation of hASCs involve transcriptional factors andsignalingmolecules. However, how these factors combinewith each other to regulate hASCs osteogenic differen-tiation still remain to be illustrated. The identification of microRNAs will illuminate this and might permit finelytuning the osteogenic differentiation process. Here, we present evidence thatmiR-218 acts as a positive regulatorof hASCs osteogenesis. Real-time PCR shows that miR-218 was up-regulated during osteogenic differentiation.Overexpression of exogenous miR-218 enhanced osteogenic differentiation in vitro, whereas inhibition of miR-218 would suppress osteogenic differentiation. Furthermore, miR-218 directly targeted SFRP2 and DKK2,which is a WNT signaling pathway antagonist, and enhanced Wnt/β-catenin signaling activity. Finally, wefound that mimicking Wnt/β-catenin signal strengthened the expression level of miR-218, while blocking thesignal attenuated the expression level of miR-218. This feed-forward regulatory circuit provides additional in-sight into howmiRNAs acting as a signal amplifier interact with signal molecules during hASCs osteogenic differ-entiation. Taken together, we have established a regulatory networkwith a central role for themiR-218 in hASCsosteogenic differentiation.

© 2013 Published by Elsevier Inc.

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RIntroduction

Human adipose-derived stem cells (hASCs) can be isolated fromadipose tissues that supply an abundant and easily accessible pool ofstem cells [1,2]. hASCs are capable of differentiating into osteogenic line-ages and have become a highly attractive source of seed cells in skeletaltissue engineering [3–5]. It has become a key issue how to effectively im-prove osteogenic differentiation of hASCs in the bone tissue engineering.

MicroRNAs (miRNAs) refer to endogenous 22-nucleotide non-codingsingle-stranded RNA molecules that suppress their targets gene expres-sion by inhibiting translation, promoting mRNA decay [6,7]. miRNAs

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egion; mRNA, messenger RNA;chain reaction; BMSCs, bone

an adipose-derived stem cells;osphatase; Runx2, runt-relatedtein 2; DKK2, dickkopf-related

, Nanjing Medical University,

ang).

vier Inc.

signal-amplification circuit btiation, Bone (2013), http://d

are involved in a broad spectrum of biologic processes, including cell dif-ferentiation, tissue development, and growth [7–9].

Recent evidence has shown that some specific miRNAs enhance orinhibit osteogenic differentiation of hASCs by targeting some inhibitorsor activators of signaling cascades [10,11]. miR-196a enhances osteo-genic differentiation of hASCs through targeting HOXC8 [10]. miR-26ainhibits osteogenic differentiation in human adipose tissue stromalcells by targeting SMAD1 [11]. However, the regulatory mechanism be-tween miRNA and osteogenic differentiation of hASCs is more similarto a network than to a linear cascade. It is still unclear howmiRNAs or-chestrate their regulation of cellular signaling and networks during os-teogenic differentiation of hASCs. The elucidation of the molecularmechanisms governing hASC osteogenic differentiation will facilitatestem cell-mediated bone regeneration therapy.

miR-218 has been found as an enhancer of bone marrow-derivedmesenchymal stem cells (BMSCs) osteogenic differentiation [12].BMSCs and ASCs possess many similar biological characteristics; how-ever, there are some differences in their differentiation potential,transcriptome and proteome [13]. Thus it is necessary to investigatethe role of miR-218 during hASC osteogenic differentiation. We foundthatmiR-218 promoted hASC osteogenic differentiation. Overexpression

etween miR-218 and Wnt/β-catenin signal promotes human adiposex.doi.org/10.1016/j.bone.2013.09.015

http://dx.doi.org/10.1016/j.bone.2013.09.015

mailto:[email protected]


http://www.sciencedirect.com/science/journal/87563282


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Table 1 t1:1

t1:2Primers used for qPCR Q3.

t1:3Gene name Real time qPCR primers (5′–3′)

t1:4Runx2 Forward: GTC TCA CTG CCT CTC ACT TGt1:5Reverse: CAC ACA TCT CCT CCC TTC TGt1:6ALP Forward: CGG ACA TCA TGA GGG TAA GGt1:7Reverse: GAG ACA TTT TCC CGT TCA CCt1:8OCN Forward: ACA GAC AAG TCC CAC ACA GCA GCt1:9Reverse: TGA AGG CTT TGT CAG ACT CAG GGCt1:10BSP Forward: GCCAGAGGAGCAATCACCAAt1:11Reverse: CAGGCTGGAGGTTCACTGGTt1:12SFRP2 Forward: CGT GGG CTC TTC CTC TTC Gt1:13Reverse: ATG TTC TGG TAC TCG ATG CCGt1:14Lef-1 Forward: AATAAAGTCCCGTGGTGCt1:15Reverse: ATGGGTAGGGTTGCCTGAATCt1:16Tcf-4 Forward: GCCTCTCATCACGTACAGCAt1:17Reverse: GGATGGGGGATTTGTCCTACt1:18miR-218 TTG TGC T TG ATC TAA CCA TGTt1:19U6 Forward: CGC TTC GGC AGC ACA TAT ACt1:20Reverse: AAA ATA TGG AAC GCT TCA CGA

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of miR-218 significantly increased hASCs osteogenic, whereas inhibitingmiR-218 had the opposite effects. Furthermore, we found that duringhASC osteogenic differentiation miR-218 directly targets 3′-UTR of theSFRP2 and DKK2 gene, which is WNT signaling pathway antagonist[14,15]. Upregulation of miR-218 decreased the expression of DKK2and SFRP2 thus activatedWnt signaling, then promoted hASC osteogenicdifferentiation; whereas inhibiting miR218 suppressed hASC osteogenicdifferentiation. MimickingWnt/β-catenin signal strengthens the expres-sion level of miR-218, while blocking the signal weakens the expressionlevel ofmiR-218. Our studies have shown a complex signal-amplificationregulatory circuit betweenmiR-218 andWnt/β-catenin signaling duringhASC osteogenic differentiation.

Materials and methods

Cell culture

All protocols involving human and animal subjects were approvedby the Ethics Committee of School of Stomatology NanjingMedical Uni-versity. Adipose tissueswere obtained frompatients undergoing tumes-cent. Bonemarrow stromal cells were isolated by flushingmarrow fromthe femurs and tibia of 6–8-weeks-old C57/BL mice. BMSCs were cul-tured as a positive control. The hASCs were isolated according to themethods as previously described [16]. To further establish that Wntsignaling could functionally affect endogenous miR-218, we examinedexpression levels of miR-218 in three biological conditions, i.e., Wnt3a,SFRP2 and DKK2 stimulated hASCs. Confluent hASCs cells were treatedwith 100 ng/ml SFRP2 (R&D), 100 ng/ml DKK2 (R&D) or 50 ng/mlWnt3a (R&D) in osteogenic medium.We used five different hASC sam-ples for each experiment and performed the duplicate experiment pereach sample.

Induction of differentiation

BMSCs or ASCswere plated at 3 × 105 cells/cm2 and cultured in com-plete culture media for 2–3 days in 6-well culture dishes. Osteogenic dif-ferentiation was induced via the culturing of the cells in osteogenicmedium (20% FBS, 0.1 μM dexamethasone, 10 mM β-glycerophosphate,and 50 μM ascorbic acid in α-MEM); the osteoblast phenotype wasevaluated by determining alkaline phosphatase (ALP) activity. The ALPactivity of hASCs was detected with a commercially available assay kit(Sigma).

Viral vector construction

Control (PMIRH000PA-1), miR-218 (PMIRH218-2PA-1) and anti-miRLenti virus (miR-Zip-218 antimiR, MZIP218-PA-1) (mature miR218sequence: 5′-TTG TGC T TG ATC TAA CCA TGT; anti miR-218: 5′-AACCACATGGTTAGATCAAGCACAA-3′; control: 5′-CCTCTTACCTCAGTTACAATTTATA-3′)were purchased from SystemBioscience. Lenti virus plasmidswere transfected by using Fugene (Promega). We used virus titers rang-ing from 5 × 105 up to 1 × 107 transducing units (TU)/ml.

miRNA extraction and qPCR analysis

Total RNA andmiRNAwere purified fromhASCs cell cultures treatedwith or without 50 μg/ml of ascorbic acid for d0, d2, d10 and d21daysusing miRNeasy mini kit (Qiagen).

Total RNA (0.5–1 μg) was reverse transcribed into cDNA using Oligo(dT) primers and was analyzed by real time qPCR. SYBR Green MasterMix (Applied Bio Systems Inc.) was used to detect the expression ofgene markers. For miRNA detection, the isolation of small species-enriched RNA was performed as per the manufacturer's instructions(mirVana miRNA isolation kit, Ambion). miRNA was reverse-transcribedwith an Ncode miRNA first-strand cDNA synthesis kit (Invitrogen)according to the manufacturer-specified guidelines. Forward primer

Please cite this article as: Zhang W-B, et al, A signal-amplification circuit btissue-derived stem cells osteogenic differentiation, Bone (2013), http://d

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sequences were designed as the corresponding mature miRNA se-quences, and U6 was used as a normalizing control. qPCR wasperformed with the Light Cycler assay, using a fluorogenic SYBR GreenI reaction mixture for PCR carried out in a Light Cycler Instrument(Roche). Data were corrected for GAPDH expression and expressed aslog (2−ΔΔCt). Primers were synthesized from Invitrogen Inc. and se-quences are listed in Table 1.

Western blot analysis

Confluent hASCs were treated under the appropriate conditions andlysed. The proteins were loaded on 10% SDS polyacrylamide gels, trans-ferred to PVDFmembrane, and probed with primary antibody for Sfrp2(Abcam), DKK2 (Abcam), actin (Santacruz), β-catenin (cell signaling),active-β-catenin (Millipore), SP1(Millipore), Axin2 (Abcam), Runx2(R&D), cyclinD1 (Abcam) and β-actin (Santacruz),. Immunoreactivebands were detected with secondary antibodies and visualized via en-hanced chemiluminescence (Amersham Pharmacia Biotech).

Nuclear and cytoplasmic fractionation

For preparation of nuclear protein, hASCs were collected andwashed twice with cold PBS. Nuclear and cytoplasmic protein wasprepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents,according to the protocols specified by the manufacturer (Pierce,Rockford, IL).

3′UTR reporter construction and reporter gene assay

The predicted consequential pairing of the target region of SFRP2,DKK2 and miR-218 sequences are in Table 2. The 3′UTRs of the SFRP2or DKK2 genes were amplified and cloned into the SacI/HindIII sites ofthe pMIR-Report luciferase vector. The seed region of the miR-218 tar-get sites in the DKK2 and SFRP2 3′UTRs were mutated using theQuickchange II site directedmutagenesis kit (Stratagene). The Luciferaseassay was conducted by co-transfecting miR-218 miRNA, NS miR andanti-miR-218 with WT and mutant pMIR-REPORT-Luc DNA construct.The transfections were performed in duplicate and all experimentswere repeated several times. Luciferase expression was normalized toβ-galactosidase activity in all cases.

Statistics

Data are expressed asmean ± SD. Student's t-test or one-way anal-ysis of variance (ANOVA) was made for multiple comparisons. All ex-periments were repeated 3 times, and representative experiments areshown. P-values b 0.05 will be considered to be statistically significant.



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Table 2t2:1

t2:2 Predicted 3′-UTR binding sites by TargetScan Human.

t2:3 Putative Target Predicted consequential pairing of target region (top) andmiRNA (bottom)

t2:4 SFRP2 SFRP2 3′UTR 5′ …ACUUCCAUUUAUUAGAAGCACAU…t2:5 hsa-miR-218 3′ UGUACCAAUCUAGUUCGUGUUt2:6 DKK2 DKK2 3′UTR 5′ …UAUUAUUAAGAGAACAAGCACAC.t2:7 hsa-miR-218 3′ UGUACCAAUCUAGUUCGUGUU

3W.-B. Zhang et al. / Bone xxx (2013) xxx–xxx

Result

Expression level of miR218 and its target genes (SFRP2 and DKK2) duringosteogenic differentiation of hASCs

To better understand the role of miR-218 in regulating the differen-tiation potential of hASCs, we investigated its expression profile duringshort- and long-term osteoblastic induction of hASCs by qPCR. Afterstandard osteoblastic induction, we determined miR-218 expressionduring the osteogenic differentiation of hASCs by qPCR. From thisstudy, we found that miR-218 expression was gradually increasedafter the induction of osteogenic differentiation of hASCs (Fig. 1A).

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Fig. 1. Expression level of miR218 and target genes (SFRP2 and DKK2) during osteogenic differetime PCR analysis of SFRP2 normalized to GAPDH during osteogenic differentiation of hASCs. C.hASCs. D. The expression of miR-218 in hASCs transfectedwith miR 218 was assayed by real-timoverexpressing miR 218. F. Real-time PCR analysis of DKK2 normalized to GAPDH by overexprassayed by real-time and normalized to U6. H. The expression of SFRP2 in hASCs transfected wmean ± SD of 3 independent experiments. **P b 0.01.


We induced hASCs to differentiate into osteoblasts by pre- or anti-miR-218. Transduced hASC were grown to confluence and osteogenicdifferentiation was induced in differentiation media.

qPCR analysis confirmed that endogenous miR-218 level was in-creased more than 300-fold compared to the control cells after twoday transfection, and remained 300-, 150-fold of the controls in 10and 21 days after pre-miR-218 infected, respectively (Fig. 1D). Thetransfected hASCs with anti-miR-218 had 4-, 3-, and 2.5-fold decreaseof miR-218 expression at 2, 10 and 21 days respectively after transfec-tion (Fig. 1G).

When miR-218 was up-regulated or down-regulated during osteo-genic differentiation, the expression levels of its targets – SFRP2, andDKK2 – also were concomitantly down-regulated or up-regulated(Figs. 1B, C, E, F, H, I).

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miR-218 promotes osteogenic differentiation of hASCs

To elucidate the impact of miR-218 on hASCs osteogenic differentia-tion,we infected hASCs by pre- or anti-miR-218. Transduced hASCweregrown to confluence and osteogenic differentiation was induced in dif-ferentiation media.

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ntiation of hASCs. A. Quantitation ofmiR-218 in indicated hASCs normalized to U6. B. Real-Real-time PCR analysis of DKK2 normalized to GAPDH during osteogenic differentiation ofe PCR and normalized to U6. E. Real-time PCR analysis of SFRP2 normalized to GAPDH by

essing miR 218. G. The expression of miR-218 in hASCs transfected with antimiR 218 wasith antimiR 218 was assayed by real-time PCR and normalized to GAPDH. The data are the



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Fig. 2A showed BMSCs' osteogenic differentiation as a positive con-trol group. Overexpression ofmiR-218 significantly increased ALP activ-ity, whereas inhibiting miR-218 had the opposite effects.

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Fig. 2.miR-218 promotes osteogenic differentiation of hASCs. A. AP staining at day 10 of BMSCsstaining at day 10 of hASCs' osteogenic differentiation by pre miR 218 and inhibitionmiR218 (mto GAPDH. D. Real-time PCR analysis of osteogenic-specific genes—OCNnormalized to GAPDH.time PCR analysis of osteogenic-specific genes — Runx2 normalized to GAPDH. The data are th


Up-regulating the expression of miR-218 obviously promotedhASCs osteogenic differentiation. Overexpression of miR-218 en-hanced osteogenic differentiation, which was indicated by the increase

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osteogenic differentiation by premiR 218 and inhibitionmiR218 (magnification 4×). B. APagnification 4×). C. Real-time PCR analysis of osteogenic-specific genes— ALP normalizedE. Real-time PCR analysis of osteogenic-specific genes— BSP normalized to GAPDH. F. Real-e mean ± SD of 3 independent experiments. **P b 0.01.



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of ALP activity (Fig. 2B). Inhibition of miR-218 has an opposite effect(Fig. 2B).

The effect was accompanied with the increased mRNA levels of sev-eral key factors that are used as osteoblastic-specific markers.

Expression of mRNAs for ALP, the osteogenic transcription factorRunx2, and the mineralization marker (bone sialoprotein (BSP) andOCN) were all significantly increased with exogenous miR-218 but de-creased with antimiR-218 (Figs. 2CDEF).

Together, these experiments demonstrate the essential role ofmiR218 for hASCs osteogenic differentiation.

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miR-218 targets Wnt inhibitors — SFRP2, DKK2 and mRNA and enhancesWnt/β-catenin signaling activity

To clarify the mechanism by which miR-218 controls hASCs osteo-genic differentiation, we focused on the identification of the potentialtargets of miR-218. To confirm the relationship between miR-218 andSFRP2/DKK2, we investigated the mRNA and protein levels of SFRP2/DKK2 in osteogenic differentiation of hASCs overexpressing miR-218or antimiR218. qPCR and Western blot analyses displayed that overex-press of miR-218 downregulated SFRP2/DKK2 expression and inhibi-tion of miR-218 upregulated SFRP2/DKK2 expression (Figs. 3A, C).

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Fig. 3. Targeting of DKK2and SFRP2 mRNA by miR-218 and regulation of Wnt signaling pathwtransfection usingmiR-C,miR-218 and anti-miR-218. Relative expressionswere normalized to Gand anti-miR-218 was analyzed by qPCR. Relative expressions were normalized to GAPDH leveWestern blot inmiR-C-,miR-218 and anti-miR-218 transfected hASCs. The protein profiles weretarget SFRP2 sequence transfected with wild type (WT) and mutated (MT) UTR constructs aloactivity was normalized with Renilla luciferase activity and expressed in relative luminescencwild type (WT) and mutated (MT) UTR constructs along with nonspecific miR control (miR-C)ciferase activity and expressed in relative luminescence units. The data represent the mean ±


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To identify a mechanism for miR-218 and Wnt signaling in hASCs,WemonitoredWnt/β-catenin signaling activity.We examinedwhethertranscriptionalmediators of activatedWnt signaling, Lef-1 and Tcf-4, re-spond to miR-218 (Fig. 3B). Over expression of miR-218 robustly in-creased endogenous Tcf-4 and Lef-1 expression, while treatment ofanti-miR-218 markedly decreased Lef-1 and Tcf-4 expression. Stimula-tion ofWnt signaling bymiR-218 through down-regulation of inhibitorsof Wnt signaling that is reflected by elevated Lef1 and Tcf4. Proteinlevels of active β-catenin and nucleus β-catenin were drastically en-hanced by miR218 treatment but decreased by anti-miR-218, concomi-tant with elevated or diminished RUNX2 as an indicator of osteoblastdifferentiation (Fig. 3C). We also examined the expression of CyclinD1 and Axin2 the classical β-catenin target gene. Cyclin D1 was in-creased by miR-218 and decreased by the anti-miR218. Axin2 was de-creased by miR-218 and increased by the anti-miR218. Axin2 is aninhibitor and well documented feedback regulator of Wnt signaling.miR-218 doesn't target Axin2 directly. miR-218 may regulate Axin2mRNA indirectly by targeting a transcription factor which inducesAxin2 [12].

A luciferase reporter assay was used to demonstrate that miR-218directly decreased DKK2 and SFRP2 expression. We aligned the miR-218 sequence with DKK2 or SFRP2 3′UTR insert and used the resultingconstruct to transfect hASCs. Transfection of miR218-infected hASCs

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ay. A. Analysis of DKK2/SFRP2 mRNA expression by real time qRT-PCR in hASCs 72 h postAPDH levels. B. Expression of Lef-1mRNA, Tcf-4mRNA inhASCs transducedwithmiR-218ls. C. SFRP2, DKK2, β-catenin, CyclinD1, Axin2 and Runx2 protein levels were analyzed bynormalizedwith anti-β-actin or anti SP1antibody. D. activity of the 3-UTR Luc reporter forng with nonspecific miR control (miR-C), miR-218, and anti-miR-218. Relative luciferasee units. E. activity of the 3-UTR Luc reporter for target DKK2 sequence transfected with, miR-218, and anti-miR-218. Relative Luciferase activity was normalized with Renilla lu-SD of 3 independent experiments. **P b 0.01.



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Fig. 4.miR218 is regulated by Wnt3a/DKK2/SFRP2. A. miR218 levels were determined inhASCs treated with Wnt3a or DKK2 or SFRP2 under osteogenic conditions. B. Real-timePCR analysis of the osteogenic gens ALP and Runx2 in hASCs treated with Wnt3a orDKK2 or SFRP2 under osteogenic conditions. C. Key transcriptional factor β-catenin andRunx2 expression levels in hASCs treatedwithWnt3a or DKK2 or SFRP2 under osteogenicconditions were analyzed byWestern blot. The data represent the mean ± SD of 3 inde-pendent experiments. **P b 0.01.


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with the parental luciferase construct (without the DKK2 or SFRP2 3′UTR) or a scrambled construct did not significantly change the expres-sion of the reporter. Cells transfected with a luciferase construct in themiR-218 target site from where the DKK2 or SFRP2 3′UTR (pMIR-DKK2 or pMIR-SFRP2) was inserted exhibited significantly lower lucif-erase activity in miR218-transfected hASCs than in control cells(Figs. 3D, E). No down regulation was observed when we mutated themiR binding sites for all potential target UTRs. Co-transfection with anantimiR218 oligo increased the luciferase activity of pMIR-DKK2 orpMIR-SFRP2 relative to co-transfection with a control oligo. These re-sults suggested that DKK2 and SFRP2 are potential direct targets ofmiR-218.

miR-218 and Wnt/β-catenin signaling are involved in a self-reinforcedregulatory circuit to regulate hASCs osteogenic differentiation

To further establish thatWnt/β-catenin signaling functionally affectsendogenous miR-218, we examined expression levels of miR-218 inthree biological conditions, i.e., Wnt3a-, DKK2 and SFRP2 stimulatedhASCs.

Our results showed that treatment of Wnt3a resulted in a nearly2.5-fold increase of miR-218 expression at 24 h. Furthermore, treat-ment of DKK-2/SFRP2 for 24 h decreased the expression of miR-218more than 2-fold.

Mimicking Wnt/β-catenin signal will strengthen the expressionlevel of miR-218, while blocking the signal weakens the expressionlevel of miR-218 (Fig. 4A). Wnt3a activated Wnt/β-catenin signal thusthe miR-218 molecule is up-regulated simultaneously and up-regulateALP, β-catenin and Runx2. DKK2/SFRP2 block Wnt/β-catenin signalthus down regulated miR-218 expression and then down regulateALP, β-catenin and Runx2 (Figs. 4B and C). Fig. 3B shows that the levelof active β-catenin proteinwas increasedwhen themiR-218 expressionwas upregulated, whereas inhibition of miR-218 leads to inhibition ofactive β-catenin. AntimiR-218 attenuated Wnt3a-induced hASC osteo-genic differentiation (Figs. 5A and B). These results have shown theassociation of miR-218 and Wnt signaling is necessary and sufficientfor osteogenesis to proceed in hASC cultures. A complex signal-amplification regulatory circuit between miR-218 and Wnt/β-cateninsignaling promotes hASC osteogenic differentiation (Fig. 6). This feed-forward regulatory circuit provides additional insight into howmiRNAsacting as a signal amplifier interact with signal molecules during hASCsosteogenic differentiation.

Discussion

Human adipose-derived stem cells (hASCs) which could differenti-ate into osteoblasts under appropriate conditions have a great applica-tion prospect in skeletal tissue engineering [3–5]. How to effectivelyimprove osteogenic differentiation of hASCs has become a key issue inthe bone tissue engineering. Osteoblast differentiation is regulated bycomplex pathways at both transcriptional and posttranscriptional levels[17,18]. MicroRNA influence osteogenic differentiation, by repressingprotein expression of their targets [18–20]. Therefore, we focused ourinvestigation of microRNA on human adipose-derived stem cells osteo-genic differentiation.

miR-218 is a potent osteo-miR expressed and continuously up-regulated during ASCs' osteogenic differentiation. The aim of this studywas to elucidate the effect and the mechanism of miR218 on hASCsosteogenic differentiation. Overexpression of miR-218 enhanced hASCsosteogenic differentiation. Inhibition ofmiR218 suppressed hASCs osteo-genic differentiation. Therefore, our data suggest thatmiR-218 positivelyregulates hASCs osteogenic differentiation.

How does miR-218 enhance hASCs' osteogensis? Our data pointstrongly to a mechanism involving the repression of Wnt antagonistsDKK2 and SFRP2, although other targets are certainly possible. miR-218overexpression downregulated the expression of DKK2 and SFRP2 at


themRNAandprotein levels. Conversely, inhibition ofmiR-218 increasedDKK2 and SFRP2 expression. These data, collected with the use of aluciferase reporter plasmid containing the DKK2/SFRP2-predicted targetgene sequences, indicated that miR-218 overexpression decreased lucif-erase activity, and inhibition of miR-218 increased luciferase activity,confirming that DKK2 and SFRP2 is a direct target of endogenous miR-218 in hASCs. Wnt signaling pathway was concordantly regulated byoverexpression and knockdown of miR-218 and has obvious link tohASCs osteogenic differentiation. Our data present evidence that miR-218 directly activates Wnt signaling promoting hASCs osteogensis. As aresult of down regulation of theDKK2/SFRP2protein level by overexpres-sion of miR-218, Wnt signaling is enhanced significantly and increasedβ-catenin transcriptional activity. Upregulation of miR-218 caused accu-mulation of active β-catenin in the cytoplasm and its translocation to thenucleus. Consequently, expression levels of Wnt target genes cyclinD1,Runx2, BSP and OCN are significantly upregulated. It is well knownthat Wnt promotes osteogenic differentiation of MSCs [21,22]. In-vivostudies have shown that constitutive active Wnt signaling leads to



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Q2 Fig. 5. AntimiR-218 attenuated Wnt3a-induced hASC osteogenic differentiation. A. ALPand Runx2 mRNA expression levels by knock down miR-218 expression in the presenceof Wnt3a treatment with hASCs. B. Active β-catenin and Runx2 expression levels byknock down miR-218 expression in the presence of Wnt3a treatment with hASCs.


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[23–25]. Conversely, deactivation of Wnt signaling by mutating LRP 5/6produces a low bonemass phenotype and inhibited osteogenic differen-tiation [14,26]. Negative regulators of Wnt signaling such as SFRPs haveall been shown to cause increased bone mass when mutated [27,28].DKK2 downregulation is a necessary in matures osteoblasts in order formineralization to proceed [27]. Recent studies also have shown thatWnt pathways are active during fracture repair and increasing the activ-ities of Wnt pathway components accelerates bone regeneration[28–31]. miR-218 modulation of hASCs osteogenic differentiation mayplan an important role in skeletal tissue regeneration. These suggestthe possibility of developing a new therapeutic approach targeting theWnt/beta-catenin pathway for improving bone regeneration whenusing hASCs as seed cells. In future work, it will be important to investi-gate whether miR-218 effectively improves hASCs' osteogensis for bone

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400401402403404405406407408409410411412413414415416417418419Fig. 6. The signal-reinforced regulatory circuit betweenmiR-218 and canonicalWnt signal.


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regeneration in vivo. This information may prove valuable in usingmiR-218 as a therapeutic targeting molecule to regulate for hASCs ap-plications in bone regeneration.

The present study has uncovered a signal amplification circuit regu-lating hASCs osteogenesis betweenmiR-218 andWnt/β-catenin signal-ing. miR-218 stimulated osteogenesis signaling by negatively regulatinginhibitors of Wnt signaling. Inhibition of Wnt/β-catenin pathwaysdecreased expression of miR-218. Activation of Wnt/β-catenin signalincreased expression of endogenous miR-218. miR-218 acts as a signal-ing amplifier for positively regulating the osteogenic differentiation ofhASCs. When osteoblastic differentiation is initiated canonical Wnt sig-nal activated. The activation of Wnt signal further enhances miR-218expression, whereas the attenuation of DKK2/SFRP2 to promote hASCsosteogenic differentiation. The signal-reinforced regulatory circuit be-tweenmiR-218 andWnt/β-catenin signal leads to the prominent effectof modulating β-catenin and Runx2 expression, which fine tunes thepace of progression in hASCs osteogenic differentiation (Fig. 6).

In conclusion, we have characterized the roles of miR-218 in osteo-genic differentiation of hASCs and elucidated the mechanisms of miR-218 action in this process. Thesefindings presentmiR-218 as a potentialtarget in the management osteogenic differentiation of hASCs for boneregeneration.

Acknowledgments

This work was supported by grants from the Natural Science Foun-dation for Colleges and Universities in JiangSu Province (Grant Number:10kJB320004), and the Priority Academic Program Development ofJiangsu Higher Education Institutions (PAPD).

Disclosure of potential conflict of interest

The authors declare no conflict of interest.

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