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Accepted Preprint first posted on 15 April 2015 as Manuscript JME-14-0314
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Glucocorticoid inhibits cell proliferation in differentiating osteoblasts by microRNA-199a targeting
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WNT signaling 1,2
Changgui Shi
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1
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1,2
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*, Ping Huang *, Hui Kang , Bo Hu , Jin Qi , Min Jiang , Hanbing Zhou , Lei 1#
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Guo , Lianfu Deng
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Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Orthopaedics and
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Traumatology, Shanghai Ruijin Hospital, Shanghai jiaotong University School of Medicine, China 2
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Department of Orthopedics, Changzheng Hospital, the Second Military Medical University of China, Shanghai, China
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* These authors contributed equally to this work:
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Changgui Shi, E-mail: [email protected]
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Ping Huang, E-mail: [email protected] #
Correspondence to:
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Dr. Lei Guo
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Professor
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Ruijin Hospital, Shanghai jiaotong University School of Medicine
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No.197, The Second Ruijin Road, Luwan District
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Shanghai, 200025, P. R. of China
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Tel: 011-86-21-643-135-34
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Fax: 011-86-21-643-357-42
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E-mail: [email protected]
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Running title: MiR-199a and osteoblasts proliferation.
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Keywords : MicroRNA-199a-5p, Glucocorticoids, WNT signaling, osteoblasts, proliferation. 1
Copyright © 2015 by the Society for Endocrinology.
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Word count of this manuscript: 4281.
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Abstract
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Inhibition of osteoblasts proliferation by glucocorticoids is very important in the etiology of
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glucocorticoid-induced osteoporosis. The mechanisms of this process are still not fully understood.
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Recent studies implicated an important role of microRNAs in glucocorticoid-mediated responses in
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various cellular processes, including cell proliferation and apoptosis. Therefore, we hypothesized that
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these regulatory molecules might be implicated in the process of glucocorticoid-decreased
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osteoblasts proliferation. Western blot, quantitative real-time PCR, cells proliferation assay, and
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luciferase assay were employed to investigate the role of microRNAs in glucocorticoid-inhibited
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osteoblasts proliferation. The microRNA-199a-5p was significantly increased in osteoblasts treated
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with dexamethasone. To delineate the role of microRNA-199a-5p, we respectively silenced and
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overexpressed microRNA-199a-5p in osteoblasts. We found that over-expressing
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microRNA-199a-5p remarkably enhanced the inhibition effect of dexamethasone on osteoblasts
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proliferation and microRNA-199a-5p depletion significantly attenuated dexamethasone-inhibited
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osteoblasts proliferation. Mechanistic studies showed that microRNA-199a-5p inhibited FZD4 and
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WNT2 expression through a microRNA-199a-5p-binding site within the 3′- untranslational region of
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FZD4 and WNT2. The post-transcriptional repression of FZD4 and WNT2 were further confirmed
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by luciferase reporter assay. These results showed that microRNA-199a-5p may play a significant
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role in the process of glucocorticoid-inhibited osteoblasts proliferation by regulating WNT signaling
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pathway.
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1 2 3 4
Introduction
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Glucocorticoids (GCs) are widely used for the treatment of inflammatory and immune diseases,
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including asthma, rheumatoid arthritis, Crohn’s disease, etc(Shi, et al. 2014; Tait, et al. 2008).
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Although GCs are used extensively to relieve these diseases, increased bone fragility attributed to
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osteopenia is a serious side effect of prolonged GCs administration(Kondo, et al. 2008). Indeed,
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GC-induced osteoporosis (GIO) is currently the third leading cause of osteoporosis following
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sex-steroid deficiency and old age(Weinstein, et al. 1998). GC-treated patients are at a twofold
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higher risk of suffering from a fracture, irrespective of their bone mineral density (BMD)(Weinstein
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2001). Limited information is available on its pathogenesis, since the clinical picture of GIO mostly
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reflects a combined effect of the underlying systemic disease and of the secondary effects induced by
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GCs treatment.
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The elucidation of the cellular and molecular mechanisms that lead to GIO and the development of
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improved means of identifying those at risk remain important challenges(Hong, et al. 2008). It is
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generally accepted that reduced bone formation is the predominant effect of GCs on bone turn
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over(Seibel, et al. 2013; Canalis, et al. 2004). Previous studies found that induction of osteoblasts
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apoptosis and inhibition of osteoblasts proliferation and differentiation finally lead to reduction of
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bone formation(Hong, et al. 2011; Pereira, et al. 2001; Weinstein, et al. 1998). It has been well
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established by both in vivo and in vitro studies that GCs regulate osteoblasts apoptosis and
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differentiation. However, the precise molecular events underlying the effect of GCs on proliferation 3
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pathways in osteoblasts are not known.
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MicroRNAs (miRNAs) are endogenous non-coding RNAs 19-25 nucleotides in length that regulate
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various biological processes including cell proliferation, apoptosis, development, hematopoiesis,
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organogenesis and tumorigenesis. The miRNAs bind to matched sequences in the 3'-untranslational
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region (3'-UTR) of target mRNAs and either repress the translation or degrade their target mRNAs
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transcript(Couzin 2007; Zamore and Haley 2005). The miRNAs may also promote translation of
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selected mRNAs in a cell cycle phase-dependent way(Zeng 2006). Recent studies implicated that
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some miRNAs play crucial role in bone formation, such as miR-27a, miR-29a, miR-34a, miR-125b,
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miR-133, miR-138, miR-199a, miR-206, miR-338, miR-335, miR-378(Taipaleenmaki, et al. 2012).
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Furthermore, previous studies confirmed that many of these miRNAs could mediate cells
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proliferation, including miR-27a, miR-29a, miR-34a, miR-125b, miR-199a, and
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miR-574(Chiyomaru, et al. 2013; Wu, et al. 2013; Huang, et al. 2012; Ma, et al. 2012; Wei, et al.
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2012; Xu, et al. 2012). Moreover, these six miRNAs were reported to regulate Wnt signaling
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pathway(Hashemi, et al. 2015; Guo, et al. 2014; Rathod, et al. 2014; Nagano, et al. 2013; Liu, et al.
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2013; Chiyomaru, et al. 2013), a crucial regulator of glucocorticoid-mediated bone acquisition and
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remodeling activities. Thus, we hypothesized that these molecules might be involved in the process
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of GC-repressed osteoblasts proliferation.
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In this study, we examined the role of miR-199a-5p in the repression of osteoblasts proliferation by
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GCs. we detected some miRNAs expression, which have been reported to be related to bone
20
formation and mediate Wnt signaling, in osteoblasts exposed to GCs. Several miRNAs were found to
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be altered, in which miR-199a-5p was identified as a strong candidate responsible for GC-decreased
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osteoblasts proliferation. Further studies confirmed that miR-199a-5p regulated osteoblasts 4
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proliferation by targeting WNT signaling. Therefore, miRNAs and miRNA-regulated gene silencing
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may contribute to inhibition effects of GCs on osteoblasts proliferation.
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Materials and Methods
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In vivo treatment of mice
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Seven-day-old neonatal C57 female mice were used for this study. Accroding to gohel et al
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report(Gohel, et al. 1999), stock solutions of 1 mg/ml Dex were prepared in ethanol. Dosing
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solutions were prepared by diluting the stock solution with normal saline. After measuring their
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weight, mice were given daily sc injections of Dex (1.0 mg/kg BW). At 72 h, mice were weighed and
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killed. The entire calvarium was removed for quantitative real-time PCR (qRT-PCR).
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Cell culture
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MC3T3-E1 cell line was supplied from Shanghai Institute of Orthopaedics and Traumatology. Lines
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< 20 passages were used in the present studies. Primary osteoblasts were obtained from neonatal
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murine calvaria using methods previously described(Shi, et al. 2014). Cells were cultured with
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alpha-minimal essential media (α-MEM) (Invitrogen, Paisley, UK) supplemented with 10% fetal
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bovine serum and 100 µg/ml penicillin/streptomycin. All experiments were performed under
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differentiation conditions, i.e. in the presence of 50 ug/ml ascorbic acid, 4 mmol/L
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beta-glycerophosphate (Sigma-Aldrich, St Louis, USA). HEK293 were cultured in Dulbecco’s
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Modified Eagle Medium (DMEM) (Invitrogen, Paisley, UK). The cultures were supplemented with
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10% fetal bovine serum and 100 µg/ml penicillin/streptomycin.
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Cell proliferation assay
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A cell counting kit (CCK-8) assay (Dojindo Laboratories, Japan) was used to measure cell
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proliferation according to the guidance of the manufacturer. Briefly, cells were resuspended in 200 µl 5
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cell culture medium and seeded at a density of 1 × 103 cells per well in 96-well microtiter plates,
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incubated overnight for cell attachment. 10 µl CCK-8 reagents were added to each well one hour
3
before the end of incubation. The optical density value (OD) of each sample was measured at a
4
wavelength of 450 nm on a microplate reader to determine the viability of the cells. The proliferation
5
rate was normalized to the value at 0 time point.
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5-ethynil-2'-deoxyuridine (EdU) assay
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Logarithmic growth phase cells were seeded in 24-well plates and incubated with serum-free
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α-MEM for 24 hours. In brief, EdU (Sigma-Aldrich, St Louis, USA) solution was added to cell
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culture medium to a final concentration of 1:1000, and then incubated for two hours. Cell fixative
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(containing 4% paraformaldehyde in PBS) was added before incubation at room temperature for 30
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minutes. After washing cells with PBS for two times, click reaction buffer (Tris-HCl, pH 8.5,
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100 mM; CuSO4, 1 mM; Apollo 550 fluorescent azide, 100 µM; ascorbic acid, 100 mM) was added
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for 10-30 min while protecting from light. Then cells were washed with 0.5% Triton X-100 for three
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times and stained subsequently with Hoechst (5 µg/ml) for 30 min at room temperature. Samples
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were stored in the dark at 4°C prior to fluorescence microscope (Olympus). EdU positive cells were
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caculated with (EdU add-in cells/Hoechst stained cells) × 100%.
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5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) assay
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Cell proliferation was measured by CFSE staining and flow cytometry(Xu, et al. 2006). Cells were
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incubated with CFSE (Invitrogen, Paisley, UK) at a concentration of 5 µmol/L in PBS for 15 minutes
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at 37℃. The reaction was stopped by adding fetal calf serum (FCS). Cells were replated at a density
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of 48,000/well in six-well dishes and incubated for 3 to 5 days. After preparation by trypsinization
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and washing, fluorescence intensity was measured on flow cytometry using excitation at 488 nm at 6
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the FL1 detection channel and analyzed with CellQuest software.
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MiR-199a-5p target-gene prediction
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We used a computation and bioinformatics-based approach to predict the putative targets of
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miR-199a-5p through TargetScan, which is hosted by the Wellcome Trust Sanger Institute. WNT
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signaling including FZD4 and WNT2 were predicted as potential target genes of miR-199a-5p by
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this program.
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Western blot analysis
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The protein samples were extracted from osteoblasts, with the procedures essentially the same as
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described in detail elsewhere(Luo, et al. 2007; Yang, et al. 2007). Protein samples (~50 µg) were
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fractionated by SDS-PAGE (7.5-10% polyacrylamide gels). Separated proteins were blot transferred
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onto a nitrocellulose membrane. After blocking with 0.1% Tween 20 and 5% nonfat dry milk in
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Tris-buffered saline at room temperature for 1 h, the membrane was incubated overnight at 4°C in
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one of the following primary antibodies: FZD4 (Peprotech, Rocky Hill, NJ) (1:400) , WNT2 (Santa
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Cruz, CA, USA) (1:400), B-catenin (Santa Cruz, CA, USA) (1:200), Runx2 (Santa Cruz, CA, USA)
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(1:200), Osterix (Santa Cruz, CA, USA) (1:400) and β-actin (Santa Cruz, CA, USA) (1:1000) as an
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internal control. The membrane was incubated with horseradish peroxidase-conjugated secondary
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antibody (1:2000) for 1 h and detected using the Enhanced Chemiluminescence (ECL) Western blot
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System (Amersham Biosciences).
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Synthesis of miRNAs and sequences of miR-199a-5p inhibitors
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MiR-199a-5p (sense: 5'-CCCAGUGUUCAGACUACCUGUUC-3' , antisense:
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5'-ACAGGUAGUCUG AACACUGGGUU-3') and its antisense oligonucleotides (AMOs:
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5'-GAACAGGTAGTCTGAACA CTGGG-3') were synthesized by Integrated DNA Technologies 7
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(IDT). Additionally, a scrambled RNA was used as negative control (NC), sense:
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5'-UUCUCCGAACGUGUCACGUTT-3' and antisense: 5'-ACGUGACACGUUCGGAGAATT-3'.
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DNA fragments of the 3'- UTRs of WNT2 and FZD4 mRNA containing the putative miR-199a-5p
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binding sequence were synthesized by Invitrogen. These fragments were then respectively cloned
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into the multiple cloning sites downstream the luciferase gene (HindIII and SpeI sites) in the
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pMIR-REPORTTM luciferase miRNA expression reporter vector (Ambion, Inc.), as described
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elsewhere(Yang, et al. 2007).
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Transfection of miR-199a-5p/AMO-199a-5p in osteoblasts.
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The MC3T3-E1 cells/primary osteoblasts were cultured with differentiation medium for 24 h.
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Osteoblasts cultured in 6-well plates was divided into different groups. Osteoblasts in the control
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group were cultured in differentiation culture medium. MicroRNA group were transfected with
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miR-199a-5p or/and inhibitors for 48 h under differentiation condition, with X-tremeGENE siRNA
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Transfection Reagent (Roche, Basel, Switzerland), as according to the manufacturer’s instructions.
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Dexamethasone (Dex) (Sigma-Aldrich, St Louis, USA) group were treated with 10 -7M Dex for 5
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days. MicroRNA together with Dex group cells were transfected with miR-199a-5p or/and inhibitors
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for 48h under 10 -7M Dex and then changed with Dex for 3 days.
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Luciferase Activity Assay
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After 24 hours starvation in serum-free medium, HEK293 cells (1-105 per well) were transfected
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with 1 µg miR-199a-5p/AMO-199a-5p or 1 µg PGL3-target DNA (firefly luciferase vector) and 0.1
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µg PRL-TK (TK-driven Renilla luciferase expression vector), with Lipofectamine 2000 (Invitrogen),
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as according to the manufacturer’s instructions. Luciferase activities were measured 48 hours after
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transfection with a dual luciferase reporter assay kit (Promega) on a luminometer (Lumat LB9507). 8
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Quantification of miRNA levels
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The mirVanaTM qRT-PCR miRNA Detection Kit (Ambion) was used in conjunction with real-time
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PCR with SYBR Green I for quantification of miR-199a-5p transcript, as detailed elsewhere(Luo, et
4
al. 2007; Yang, et al. 2007).
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Statistics
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All data were analysed by using the SPSS 19.0 Software (SPSS, Inc.). The composite data are
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expressed as means ± s.e.m. Statistical analysis was performed with one-way ANOVA followed by
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Dunnett’s test where appropriate. Differences were considered to be significant at P≤0.05.
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Results
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The effect of Dex on differentiating osteoblasts proliferation.
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To determine whether Dex regulated the proliferation of differentiating osteoblasts, we firstly used
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CCK-8 assay to monitor cell proliferation. MC3T3-E1 cells under differentiation conditions were
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treated with Dex at the concentrations (0, 10–9, 10–8, 10–7 M) from day 1 to day 5. Our results showed
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that Dex decreased the proliferation of MC3T3-E1 cells in a dose- and time- dependent manner
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(Figure 1A).
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Furthermore, MC3T3-E1 cells were stimulated with 10–7 M Dex for 5 days prior to EdU assay.
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EdU-stained photomicrographs and corresponding photomicrographs of total cells were shown in
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Figure 1B. The proportion of cells with EdU-positive nuclei was shown in Figure 1C. EdU
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incorporation was decreased in the Dex group, indicating that Dex could inhibit the MC3T3-E1 cells
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proliferation.
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To further examine proliferation, MC3T3-E1cells were cultured with 10–7 M Dex for 5 days and
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stained with CFSE. CFSE irreversibly couples to cellular proteins. When cells divide, CFSE labeling 9
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is distributed equally between daughter cells, which are half as fluorescent as their parents. The peak
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CFSE fluorescence intensity on flow cytometry was shifted by Dex, indicating that cells treated with
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Dex had fewer cycles of cell replication as compared with the control group (Figure 1D).
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Up-regulation of miR-199a-5p expression by Dex in differentiating osteoblasts.
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To examine the role of miRNAs in the repression of osteoblasts proliferation by Dex, we firstly
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detected some miRNAs expression, which have been reported to be related to bone formation and
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mediate WNT signaling, in MC3T3-E1 cells exposed to 10–7 M Dex for 5 days. Several miRNAs
8
were found to be altered, in which miR-199a-5p was identified as a strong candidate responsible for
9
GC-decreased osteoblasts proliferation. We found that miR-574-3p and miR-27a were weakly
10
affected during this time frame. However, the expression of miR-199a-5p was significantly
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upregulated by Dex (Figure 2A). Further studies demonstrated that Dex increased the expression of
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miR-199a-5p in a time- dependent manner (Figure 2B). Similarly, the upregulation of miR-199a-5p
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expression was also observed in primary osteoblasts treated with 10-7 M Dex for 5 days (Figure 2C).
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Furthermore, we observed the effects of Dex on miR-199a-5p expression in vivo. We found that the
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expression of miR-199a-5p was significantly upregulated in calvarias from mice treated with Dex
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(Figure 2D). Thus, we hypothesized that miR-199a-5p might be involved in the process of
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Dex-decreased osteoblasts proliferation.
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Repression of differentiating osteoblasts proliferation by miR-199a-5p.
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To delineate the role of miR-199a-5p in osteoblasts proliferation, we performed loss-of-function and
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gain-of-function experiments in which we decreased and increased the quantities of miR-199a-5p
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with miR-199a-5p inhibitor and miR-199a-5p mimic, respectively. MC3T3-E1 cells were transfected
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with miR-199a-5p or/and AMO-199a-5p from day 1 to day 5. Then we assessed cell proliferation 10
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with CCK-8 assays. We found that overexpression of miR-199a-5p decreased MC3T3-E1 cells
2
proliferation. However, the depletion of miR-199a-5p with AMO-199a-5p resulted in the increased
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MC3T3-E1 cells proliferation.
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Furthermore, EdU assay by fluorescence microscope further demonstrated that miR-199a-5p mimic
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clearly decreased MC3T3-E1 cells proliferation (Figure 3B,C), and miR-199a-5p inhibitor
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significantly enhanced osteoblasts proliferation. Similar results were further confirmed by CFSE
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fluorescence intensity assayment (Figure 3D). In addition, the effect of miR-199a-5p on osteoblasts
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differentiation was observed by western blot. The expressions of osteogenic marker genes including
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Runx2 and Osterix were significantly affected in MC3T3-E1 cells transfected with miR-199a-5p
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or/and AMO-199a-5p for 48h, suggesting that miR-199a-5p was also involved in the process of
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osteoblasts differentiation (Figure 3E).
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Furthermore, primary osteoblasts were also used to test the effects of miR-199a-5p on cells
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proliferation. The proliferation of primary osteoblasts transfected with miR-199a-5p or/and
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AMO-199a-5p was measured by CCK8, EdU assay and CFSE fluorescence intensity assayment.
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Similarly, we found that miR-199a-5p significantly decreased primary osteoblasts proliferation and
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AMO-199a-5p enhanced it (Figure 4A-C).
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could regulate osteoblasts proliferation. Involvement of miR-199a-5p in Dex-reduced osteoblasts
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proliferation.
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We consequently investigated whether miR-199a-5p was involved in Dex-decreased osteoblasts
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proliferation. To this end, we observed the effect of Dex on proliferation in MC3T3-E1 cells
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transfected with miR-199a-5p or/and AMO-199a-5p for 5 days. CCK-8 assay showed that
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miR-199a-5p markedly enhanced Dex-decreased MC3T3-E1 cells proliferation. Furthermore, 11
Taken together, these results indicated that miR-199a-5p
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co-application of miR-199a-5p and AMO-199a-5p almost completely abolished the effect of
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miR-199a-5p. Moreover, the inhibitory effect of Dex on MC3T3-E1 cells proliferation was
3
significantly alleviated when MC3T3-E1 cells were alone transfected with AMO-199a-5p, indicating
4
that miR-199a-5p was involved in Dex-decreased osteoblasts proliferation (Figure 5A). Similar
5
results were further confirmed by EdU assay and flow cytometric analysis of CFSE intensity (Figure
6
5B-C). All of above results were further verified in primary osteoblasts (Figure 6A-C).
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Repression of WNT signaling by miR-199a-5p transfection.
8
Based on the above observations, miR-199a-5p was involved in osteoblasts proliferation. It is
9
possible that miR-199a-5p targets several regulatory factors related to osteoblasts proliferation. To
10
address this issue, we used a computation and bioinformatics-based approach to predict the putative
11
targets related to proliferation through TargetScan, which is hosted by the Wellcome Trust Sanger
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Institute. These explorations lead to the identification of candidate targets of miR-199a-5p: WNT
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signaling including FZD4 and WNT2 (Figure 7A,B). Western blot analysis of FZD4 and WNT2
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expression in the MC3T3-E1 cells treated with 10-7 M Dex for 5 days are showed in Figure 7C,D.
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We found that Dex decreased FZD4 and WNT2 expression in MC3T3-E1 cells. To prove that FZD4
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and WNT2 are indeed repressed posttranscriptionally by miR-199a-5p, we determined the effect of
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the miR-199a-5p on protein expression. Western blot analysis showed that miR-199a-5p lowered
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markedly the levels of FZD4 and WNT2 proteins in MC3T3-E1 (Figure 7E,F). Co-application of
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miR-199a-5p and AMO-199a-5p abolished almost completely the effect of miR-199a-5p. Moreover,
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application of the AMO-199a-5p alone increased the levels of FZD4 and WNT2 in MC3T3-E1,
21
indicating that there is a basal level of miR-199a-5p activity in osteoblasts (Figure 7E,F). Similarly,
22
we also test the effects of miR-199a-5p on the levels of FZD4 and WNT2 proteins in primary 12
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osteoblasts. Our results showed that miR-199a-5p significantly decreased FZD4 and WNT2 proteins
2
expression and AMO-199a-5p increased it (Figure 8A,B). In addition, we observed the effect of
3
miR-199a-5p on the expression of B-catenin, a key mediater of WNT signaling pathway, in primary
4
osteoblasts. We found that miR-199a-5p decreased B-catenin expression, suggesting that
5
miR-199a-5p could inhibit WNT signaling pathway (Figure 8B).
6
Verification of interactions between miR-199a-5p and their target genes.
7
We constructed chimeric vectors by placing the 3'-UTRs of FZD4 and WNT2 into the 3'-UTR of a
8
luciferase reporter plasmid. We performed luciferase reporter assays in HEK293 cells that do not
9
express miR-199a-5p (Data not shown). Compared with the NC, transfection of miR-199a-5p with
10
the luciferase reporter gene linked to the 3'-UTR of FZD4 or WNT2 resulted in a significant decrease
11
of luciferase activity, and co-application of miR-199a-5p with AMO-199a-5p alleviated the decrease
12
of luciferase activity, whereas AMO-199a-5p alone had no effect (Figure 9A,B). These data suggest
13
that FZD4 and WNT2 are the targets of miR-199a-5p.
14
Successful delivery of miR-199a-5p, AMO-199a-5p and NC to the cells were further verified by
15
comparing the miR-199a-5p levels 48 hours after transfection of the constructs in MC3T3-E1 cells
16
and primary osteoblasts. Transfection resulted in significant increasing in miR-199a-5p levels
17
(Figure 9C). It is worth noting that the miR-199a-5P levels were dynamic after transfection. Our data
18
were collected at a specific time which was 48 hours after transfection, because the plateau level was
19
reached then. These results proved the feasibility of all experiments.
20
Discussion
21
Increased concentrations of GCs could cause the development of Cushing’s syndrome, with severe
22
osteoporosis. Previous studies have revealed that GCs have potent inhibitory effects on osteoblasts 13
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proliferation(Hong, et al. 2011; Walsh, et al. 2001). However, the molecular mechanisms involved in
2
the GC-inhibited osteoblasts proliferation are still poorly understood. In this study, we have
3
illustrated that miR-199a-5p plays a significant role in the process of GC-decreased osteoblasts
4
proliferation, by regulating WNT signaling pathway.
5
Several recent reports have suggested that GCs exert post-transcriptional control through the
6
regulation of miRNAs processing and expression(De Iudicibus, et al. 2013). Xing et al.reported that
7
GCs induced apoptosis by inhibiting miRNAs cluster miR-17~92 expression in chondrocytic
8
cells(Xing, et al. 2014). Furthermore, miR-29b and miR-29c were reported to be involved in
9
Toll-like receptor control of GC-induced apoptosis in human plasmacytoid dendritic cells(Hong, et al.
10
2013). In addition, miRNAs could also negatively regulate the GC receptor (GR) transcriptional
11
response by directly targeting the 3'-UTR of GRα mRNA(Lv, et al. 2012). It is also known that
12
osteoblasts are critical target cells of GCs. Our previous studies showed that miR-17~92a
13
downregulation by GCs leads to Bim targeting and induction of osteoblasts apoptosis(Guo, et al.
14
2013). Furthermore, GCs could also increase Receptor activator of nuclear factor B ligand (RANKL)
15
expression by down-regulation of miR-17/20a in osteoblasts, which indirectly enhances
16
osteoclastogenesis and bone resorption(Shi, et al. 2014). However, to our knowledge, there have
17
been no reports on whether miRNAs expression could be regulated by GCs in osteoblasts
18
proliferation. The present study is the first effort to observe the effect of GCs on miRNAs in
19
osteoblasts proliferation.
20
In this study, we detected some miRNAs expression in osteoblasts exposed to GCs. These miRNAs
21
have been reported to be related to cell proliferation and bone formation in previous
22
studies(Chiyomaru, et al. 2013; Wu, et al. 2013; Taipaleenmaki, et al. 2012; Huang, et al. 2012; Ma, 14
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et al. 2012; Wei, et al. 2012; Xu, et al. 2012). Furthermore, these miRNAs could mediate WNT
2
signaling pathway(Hashemi, et al. 2015; Guo, et al. 2014; Rathod, et al. 2014; Nagano, et al. 2013;
3
Liu, et al. 2013; Chiyomaru, et al. 2013), a curcial factor of GC-decreased bone formation. Therefore,
4
we firstly examined whether these six miRNAs were involved in the regulation of osteoblasts
5
proliferation by GC. Our results showed that only miR-199a-5p was identified as a strong candidate
6
responsible for GC-decreased osteoblasts proliferation. We speculated that miRNAs might play
7
different roles in different cells. Our study did not apply miRNAs chip to screen miRNAs expression
8
in response to GC-decreased osteoblasts proliferation. Therefore, several differentially expressed
9
miRNAs may be missed. Future studies are required to identify other miRNAs involved in
10
GC-inhibited osteoblasts proliferation.
11
Our results identified miR-199a-5p was significantly increased during Dex-decreased osteoblasts
12
proliferation. Furthermore, overexpression of miR-199a-5p decreased osteoblasts proliferation.
13
However, the depletion of miR-199a-5p with AMO-199a-5p resulted in the increased proliferation of
14
osteoblasts. Previous studies showed that miR-199a-5p was a ubiquitously expressed miRNA, whose
15
expression was modulated at key time points of development, growth, regeneration, cancers(Shi, et
16
al. 2014; Alexander, et al. 2013; Xu, et al. 2012). Shi et al. showed that miR-199a-5p affected
17
porcine preadipocyte proliferation and differentiation(Shi, et al. 2014). Alexander et al. found that
18
miR-199a-5p affected WNT signaling, cell proliferation, and myogenic differentiation(Alexander, et
19
al. 2013). Furthermore, Lentivirus-mediated overexpression of miR-199a-5p inhibited cell
20
proliferation of human hepatocellular carcinoma(Xu, et al. 2012). Although these studies have
21
reported the role of miR-199a-5p in the pathogenesis of several diseases, including breast cancer,
22
lymphoma or pulmonary hypertension, our study is the first report to uncover the role of 15
Page 16 of 36
1
miR-199a-5p in metabolic bone diseases and osteoblasts proliferation.
2
GCs, which regulate diverse physiological effects, have established both genomic and nongenomic
3
mechanisms(Stahn and Buttgereit 2008). The steroid’s nongenomic effects occur within seconds to
4
minutes and are mediated by the GR or by other means such as G protein-coupled receptors(Stahn
5
and Buttgereit 2008). It has been elucidated that GCs are unable to suppress bone formation in the
6
absence of GR expression in osteoblasts as their proliferation, differentiation, and apoptosis will
7
become immune to GCs(Rauch, et al. 2010). Therefore, GC-inhibited proliferation of osteoblasts is
8
triggered via GR-dependent transcriptional regulation. Previous study reported that GCs could
9
regulate miRNAs expression via a GR-mediated direct DNA binding mechanism(De Iudicibus, et al.
10
2013). In this study, we did not explore whether GR was involved in the regulation of miR-199a-5p
11
by GCs in osteoblasts. Therefore, further studies need to be done to uncover the progress of
12
GC-regulated miR-199a-5p in osteoblasts. WNT/ B-catenin signaling plays an important role in bone
13
development and metabolism by controlling both bone formation and resorption(Wang, et al. 2014).
14
Wnt ligands are secreted molecules that bind to cell surface receptors encoded by the Frizzled and
15
low-density lipoprotein receptor-related proteins (LRPs). Once bound, the ligands initiate a cascade
16
of intracellular events that activate the B-catenin activity and the DNA binding protein TCF, which
17
eventually result in the transcription of target genes(Wang, et al. 2014). It has been confirmed that
18
GCs could inhibit canonical WNT signaling to suppress bone formation(Guanabens, et al. 2014). In
19
this study, miR-199a-5p was involved in GC-inhibited osteoblasts proliferation through targeting
20
WNT signaling. Previous studies also showed that miR-199a-5p could target several cell
21
proliferation regulatory factors within the WNT signaling pathway, including FZD4 and
22
WNT2(Alexander, et al. 2013). 16
Page 17 of 36
1
Our data provide new evidence that miR-199a-5p plays a dominant effect in GC-inhibited
2
osteoblasts proliferation. The inhibitory effect of miR-199a-5p on osteoblasts proliferation is
3
ascribed to the disrupting of WNT signaling. This study is an effort to better understand the
4
molecular mechanism of GIO, and to provide new insights into the potential contribution of miRNAs
5
in the process of GC-mediated osteoblasts proliferation and bone formation.
6
Declaration of interest
7
The authors declare that there is no conflict of interest that could be perceived as prejudicing the
8
impartiality of the research reported.
9
Funding
10
This work was supported by the National Natural Science Foundation of China [Grant number.
11
81300713]; Innovation Program of Shanghai Municipal Education Commision [Grant number.
12
14YZ044]; and the Shanghai Jiaotong University School of Medicine Grant [Grant number.
13
13XJ10060].
14
Author contributions
15
L.G. and C.G.S. were involved in the conception and hypothesis delineation; L.G. and C.G.S.
16
designed the experiments, conducted the luciferase and quantitative real-time PCR experiments, and
17
wrote the article; J.Q., B.H., H.B.Z. and H.K. performed EdU staining and CFSE assay; P.H.,
18
modified the manuscript; M.J. and P.H. performed a part of the luciferase, western blot analysis;
19
L.F.D. designed and conducted the animal studies.
20
Acknowledgements
21
The authors thank Dr Zhaoping Qiu for expert technical assistance in carrying out Real time RT-PCR
22
and western blotting analysis. 17
Page 18 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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Figure legends
2
Figure 1. The inhibition of differentiating osteoblasts proliferation by Dex. A: Proliferation of MC3T3-E1
3
cells was measured by CCK8 after cells were treated with 10–9, 10–8, 10–7 M Dex from day 1 to day 5. The
4
results showed that Dex decreased the viability of MC3T3-E1 cells in a dose- and time- dependent manner.
5
n = 3, **P < 0.01. B: Representative photomicrographs of EdU staining (top panel) and corresponding total
6
cell photomicrographs (middle panel). Blue: Hoechst labeling of cell nuclei; Red: EdU labeling of nuclei of
7
proliferative cells (×400). C: Quantitative data showing the percentage of EdU-positive cells in different
8
treatment groups (number of red vs number of blue nuclei). n = 3, **P < 0.01. D: MC3T3-E1 cells were
9
stained with CFSE before plating, cultured for 5 days, and analyzed by flow cytometry as described in
10
Materials and Methods. The peak CFSE fluorescence intensity on flow cytometry was shifted by Dex. n = 3.
11
Con indicates control, Dex indicates dexamethasone.
12
Figure 2. Up-regulation of miR-199a-5p expression by Dex in differentiating osteoblasts. A: qRT-PCR
13
analysis of miR-574-3p, miR-27a, miR-29a, miR-34a, miR-125b and miR-199a-5p expression in MC3T3-
14
E1 cells treated with 10-7 M Dex for 5 days. n = 3, **P < 0.01. B: qRT-PCR analysis of miR-199a-5p
15
expression in MC3T3-E1 cells treated with 10–7 M Dex from day 1 to day 5. n = 3, **P < 0.01. C: qRT-
16
PCR analysis of miR-199a-5p expression in primary osteoblasts treated with 10–7 M Dex for 5 days. n = 3,
17
**P < 0.01. D: qRT-PCR analysis of miR-199a-5p expression in the calvarias from mice treated with Dex
18
for 3 days. n = 3, **P < 0.01. Con indicates control, Dex indicates dexamethasone, miR indicates
19
microRNA.
20
Figure 3. Repression of MC3T3-E1 cells proliferation by miR-199a-5p. A: Proliferation of MC3T3-E1 cells
21
was measured by CCK8 after cells were transfected with miR-199a-5p or/and AMO-199a-5p from day 1 to
22
day 5. n = 3, **P < 0.01. B: Representative photomicrographs of EdU staining (top panel) and
23
corresponding total cell photomicrographs (middle panel). Blue: Hochest labeling of cell nuclei; Red: EdU 1
Page 25 of 36 1
labeling of nuclei of proliferative cells (×400). C: Quantitative data showing the percentage of EdU-positive
2
cells in different treatment groups (number of red vs number of blue nuclei). n = 3, **P < 0.01. D: MC3T3-
3
E1 cells transfected with miR-199a-5p or/and AMO-199a-5p were stained with CFSE and analyzed by flow
4
cytometry as described in Materials and Methods. n = 3. E: Western blot analysis of Runx2 and Osterix
5
expression in MC3T3-E1 cells transfected with miR-199a-5p or/and AMO-199a-5p. n = 3, **P < 0.01, *P
6
< 0.05. Con indicates control, NC indicates negative control, miR indicates microRNA.
7
Figure 4. Repression of primary osteoblasts proliferation by miR-199a-5p. A: Proliferation of primary
8
osteoblasts was measured by CCK8 after cells were transfected with miR-199a-5p or/and AMO-199a-5p
9
from day 1 to day 5. n = 3, **P < 0.01. B: Quantitative data showing the percentage of EdU-positive cells in
10
different treatment groups (number of red vs number of blue nuclei). n = 3, **P < 0.01, *P < 0.05. C:
11
Primary osteoblasts transfected with miR-199a-5p or/and AMO-199a-5p were stained with CFSE and
12
analyzed by flow cytometry as described in Materials and Methods. n = 3. Con indicates control, NC
13
indicates negative control, miR indicates microRNA.
14
Figure 5. Involvement of miR-199a-5p in Dex-reduced MC3T3-E1 cells proliferation. A: Proliferation of
15
MC3T3-E1 cells was measured by CCK8 after cells were transfected with miR-199a-5p or/and AMO-199a-
16
5p under Dex from day 1 to day 5. n = 3, **P < 0.01. B: Quantitative data showing the percentage of EdU-
17
positive cells in different treatment groups (number of red vs number of blue nuclei). n = 3, **P < 0.01. C:
18
MC3T3-E1 cells transfected with miR-199a-5p or/and AMO-199a-5p under Dex were stained with CFSE
19
and analyzed by flow cytometry as described in Materials and Methods. n = 3. Con indicates control, Dex
20
indicates dexamethasone, NC indicates negative control, miR indicates microRNA.
21
Figure 6. Involvement of miR-199a-5p in Dex-reduced primary osteoblasts proliferation. A: Proliferation of
22
primary osteoblasts was measured by CCK8 after cells were transfected with miR-199a-5p or/and AMO-
23
199a-5p under Dex from day 1 to day 5. n = 3, **P < 0.01. B: Quantitative data showing the percentage of 2
Page 26 of 36 1
EdU-positive cells in different treatment groups (number of red vs number of blue nuclei). n = 3, **P
Accepted Preprint first posted on 15 April 2015 as Manuscript JME-14-0314
1
Glucocorticoid inhibits cell proliferation in differentiating osteoblasts by microRNA-199a targeting
2
WNT signaling 1,2
Changgui Shi
3
1
1
1,2
1
1
1
*, Ping Huang *, Hui Kang , Bo Hu , Jin Qi , Min Jiang , Hanbing Zhou , Lei 1#
1
Guo , Lianfu Deng
4 1
5
Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Orthopaedics and
6
Traumatology, Shanghai Ruijin Hospital, Shanghai jiaotong University School of Medicine, China 2
7
Department of Orthopedics, Changzheng Hospital, the Second Military Medical University of China, Shanghai, China
8 9
* These authors contributed equally to this work:
10
Changgui Shi, E-mail: [email protected]
11
Ping Huang, E-mail: [email protected] #
Correspondence to:
12 13
Dr. Lei Guo
14
Professor
15
Ruijin Hospital, Shanghai jiaotong University School of Medicine
16
No.197, The Second Ruijin Road, Luwan District
17
Shanghai, 200025, P. R. of China
18
Tel: 011-86-21-643-135-34
19
Fax: 011-86-21-643-357-42
20
E-mail: [email protected]
21
Running title: MiR-199a and osteoblasts proliferation.
22
Keywords : MicroRNA-199a-5p, Glucocorticoids, WNT signaling, osteoblasts, proliferation. 1
Copyright © 2015 by the Society for Endocrinology.
Page 2 of 36
1
Word count of this manuscript: 4281.
2
Abstract
3
Inhibition of osteoblasts proliferation by glucocorticoids is very important in the etiology of
4
glucocorticoid-induced osteoporosis. The mechanisms of this process are still not fully understood.
5
Recent studies implicated an important role of microRNAs in glucocorticoid-mediated responses in
6
various cellular processes, including cell proliferation and apoptosis. Therefore, we hypothesized that
7
these regulatory molecules might be implicated in the process of glucocorticoid-decreased
8
osteoblasts proliferation. Western blot, quantitative real-time PCR, cells proliferation assay, and
9
luciferase assay were employed to investigate the role of microRNAs in glucocorticoid-inhibited
10
osteoblasts proliferation. The microRNA-199a-5p was significantly increased in osteoblasts treated
11
with dexamethasone. To delineate the role of microRNA-199a-5p, we respectively silenced and
12
overexpressed microRNA-199a-5p in osteoblasts. We found that over-expressing
13
microRNA-199a-5p remarkably enhanced the inhibition effect of dexamethasone on osteoblasts
14
proliferation and microRNA-199a-5p depletion significantly attenuated dexamethasone-inhibited
15
osteoblasts proliferation. Mechanistic studies showed that microRNA-199a-5p inhibited FZD4 and
16
WNT2 expression through a microRNA-199a-5p-binding site within the 3′- untranslational region of
17
FZD4 and WNT2. The post-transcriptional repression of FZD4 and WNT2 were further confirmed
18
by luciferase reporter assay. These results showed that microRNA-199a-5p may play a significant
19
role in the process of glucocorticoid-inhibited osteoblasts proliferation by regulating WNT signaling
20
pathway.
21 22 2
Page 3 of 36
1 2 3 4
Introduction
5
Glucocorticoids (GCs) are widely used for the treatment of inflammatory and immune diseases,
6
including asthma, rheumatoid arthritis, Crohn’s disease, etc(Shi, et al. 2014; Tait, et al. 2008).
7
Although GCs are used extensively to relieve these diseases, increased bone fragility attributed to
8
osteopenia is a serious side effect of prolonged GCs administration(Kondo, et al. 2008). Indeed,
9
GC-induced osteoporosis (GIO) is currently the third leading cause of osteoporosis following
10
sex-steroid deficiency and old age(Weinstein, et al. 1998). GC-treated patients are at a twofold
11
higher risk of suffering from a fracture, irrespective of their bone mineral density (BMD)(Weinstein
12
2001). Limited information is available on its pathogenesis, since the clinical picture of GIO mostly
13
reflects a combined effect of the underlying systemic disease and of the secondary effects induced by
14
GCs treatment.
15
The elucidation of the cellular and molecular mechanisms that lead to GIO and the development of
16
improved means of identifying those at risk remain important challenges(Hong, et al. 2008). It is
17
generally accepted that reduced bone formation is the predominant effect of GCs on bone turn
18
over(Seibel, et al. 2013; Canalis, et al. 2004). Previous studies found that induction of osteoblasts
19
apoptosis and inhibition of osteoblasts proliferation and differentiation finally lead to reduction of
20
bone formation(Hong, et al. 2011; Pereira, et al. 2001; Weinstein, et al. 1998). It has been well
21
established by both in vivo and in vitro studies that GCs regulate osteoblasts apoptosis and
22
differentiation. However, the precise molecular events underlying the effect of GCs on proliferation 3
Page 4 of 36
1
pathways in osteoblasts are not known.
2
MicroRNAs (miRNAs) are endogenous non-coding RNAs 19-25 nucleotides in length that regulate
3
various biological processes including cell proliferation, apoptosis, development, hematopoiesis,
4
organogenesis and tumorigenesis. The miRNAs bind to matched sequences in the 3'-untranslational
5
region (3'-UTR) of target mRNAs and either repress the translation or degrade their target mRNAs
6
transcript(Couzin 2007; Zamore and Haley 2005). The miRNAs may also promote translation of
7
selected mRNAs in a cell cycle phase-dependent way(Zeng 2006). Recent studies implicated that
8
some miRNAs play crucial role in bone formation, such as miR-27a, miR-29a, miR-34a, miR-125b,
9
miR-133, miR-138, miR-199a, miR-206, miR-338, miR-335, miR-378(Taipaleenmaki, et al. 2012).
10
Furthermore, previous studies confirmed that many of these miRNAs could mediate cells
11
proliferation, including miR-27a, miR-29a, miR-34a, miR-125b, miR-199a, and
12
miR-574(Chiyomaru, et al. 2013; Wu, et al. 2013; Huang, et al. 2012; Ma, et al. 2012; Wei, et al.
13
2012; Xu, et al. 2012). Moreover, these six miRNAs were reported to regulate Wnt signaling
14
pathway(Hashemi, et al. 2015; Guo, et al. 2014; Rathod, et al. 2014; Nagano, et al. 2013; Liu, et al.
15
2013; Chiyomaru, et al. 2013), a crucial regulator of glucocorticoid-mediated bone acquisition and
16
remodeling activities. Thus, we hypothesized that these molecules might be involved in the process
17
of GC-repressed osteoblasts proliferation.
18
In this study, we examined the role of miR-199a-5p in the repression of osteoblasts proliferation by
19
GCs. we detected some miRNAs expression, which have been reported to be related to bone
20
formation and mediate Wnt signaling, in osteoblasts exposed to GCs. Several miRNAs were found to
21
be altered, in which miR-199a-5p was identified as a strong candidate responsible for GC-decreased
22
osteoblasts proliferation. Further studies confirmed that miR-199a-5p regulated osteoblasts 4
Page 5 of 36
1
proliferation by targeting WNT signaling. Therefore, miRNAs and miRNA-regulated gene silencing
2
may contribute to inhibition effects of GCs on osteoblasts proliferation.
3
Materials and Methods
4
In vivo treatment of mice
5
Seven-day-old neonatal C57 female mice were used for this study. Accroding to gohel et al
6
report(Gohel, et al. 1999), stock solutions of 1 mg/ml Dex were prepared in ethanol. Dosing
7
solutions were prepared by diluting the stock solution with normal saline. After measuring their
8
weight, mice were given daily sc injections of Dex (1.0 mg/kg BW). At 72 h, mice were weighed and
9
killed. The entire calvarium was removed for quantitative real-time PCR (qRT-PCR).
10
Cell culture
11
MC3T3-E1 cell line was supplied from Shanghai Institute of Orthopaedics and Traumatology. Lines
12
< 20 passages were used in the present studies. Primary osteoblasts were obtained from neonatal
13
murine calvaria using methods previously described(Shi, et al. 2014). Cells were cultured with
14
alpha-minimal essential media (α-MEM) (Invitrogen, Paisley, UK) supplemented with 10% fetal
15
bovine serum and 100 µg/ml penicillin/streptomycin. All experiments were performed under
16
differentiation conditions, i.e. in the presence of 50 ug/ml ascorbic acid, 4 mmol/L
17
beta-glycerophosphate (Sigma-Aldrich, St Louis, USA). HEK293 were cultured in Dulbecco’s
18
Modified Eagle Medium (DMEM) (Invitrogen, Paisley, UK). The cultures were supplemented with
19
10% fetal bovine serum and 100 µg/ml penicillin/streptomycin.
20
Cell proliferation assay
21
A cell counting kit (CCK-8) assay (Dojindo Laboratories, Japan) was used to measure cell
22
proliferation according to the guidance of the manufacturer. Briefly, cells were resuspended in 200 µl 5
Page 6 of 36
1
cell culture medium and seeded at a density of 1 × 103 cells per well in 96-well microtiter plates,
2
incubated overnight for cell attachment. 10 µl CCK-8 reagents were added to each well one hour
3
before the end of incubation. The optical density value (OD) of each sample was measured at a
4
wavelength of 450 nm on a microplate reader to determine the viability of the cells. The proliferation
5
rate was normalized to the value at 0 time point.
6
5-ethynil-2'-deoxyuridine (EdU) assay
7
Logarithmic growth phase cells were seeded in 24-well plates and incubated with serum-free
8
α-MEM for 24 hours. In brief, EdU (Sigma-Aldrich, St Louis, USA) solution was added to cell
9
culture medium to a final concentration of 1:1000, and then incubated for two hours. Cell fixative
10
(containing 4% paraformaldehyde in PBS) was added before incubation at room temperature for 30
11
minutes. After washing cells with PBS for two times, click reaction buffer (Tris-HCl, pH 8.5,
12
100 mM; CuSO4, 1 mM; Apollo 550 fluorescent azide, 100 µM; ascorbic acid, 100 mM) was added
13
for 10-30 min while protecting from light. Then cells were washed with 0.5% Triton X-100 for three
14
times and stained subsequently with Hoechst (5 µg/ml) for 30 min at room temperature. Samples
15
were stored in the dark at 4°C prior to fluorescence microscope (Olympus). EdU positive cells were
16
caculated with (EdU add-in cells/Hoechst stained cells) × 100%.
17
5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) assay
18
Cell proliferation was measured by CFSE staining and flow cytometry(Xu, et al. 2006). Cells were
19
incubated with CFSE (Invitrogen, Paisley, UK) at a concentration of 5 µmol/L in PBS for 15 minutes
20
at 37℃. The reaction was stopped by adding fetal calf serum (FCS). Cells were replated at a density
21
of 48,000/well in six-well dishes and incubated for 3 to 5 days. After preparation by trypsinization
22
and washing, fluorescence intensity was measured on flow cytometry using excitation at 488 nm at 6
Page 7 of 36
1
the FL1 detection channel and analyzed with CellQuest software.
2
MiR-199a-5p target-gene prediction
3
We used a computation and bioinformatics-based approach to predict the putative targets of
4
miR-199a-5p through TargetScan, which is hosted by the Wellcome Trust Sanger Institute. WNT
5
signaling including FZD4 and WNT2 were predicted as potential target genes of miR-199a-5p by
6
this program.
7
Western blot analysis
8
The protein samples were extracted from osteoblasts, with the procedures essentially the same as
9
described in detail elsewhere(Luo, et al. 2007; Yang, et al. 2007). Protein samples (~50 µg) were
10
fractionated by SDS-PAGE (7.5-10% polyacrylamide gels). Separated proteins were blot transferred
11
onto a nitrocellulose membrane. After blocking with 0.1% Tween 20 and 5% nonfat dry milk in
12
Tris-buffered saline at room temperature for 1 h, the membrane was incubated overnight at 4°C in
13
one of the following primary antibodies: FZD4 (Peprotech, Rocky Hill, NJ) (1:400) , WNT2 (Santa
14
Cruz, CA, USA) (1:400), B-catenin (Santa Cruz, CA, USA) (1:200), Runx2 (Santa Cruz, CA, USA)
15
(1:200), Osterix (Santa Cruz, CA, USA) (1:400) and β-actin (Santa Cruz, CA, USA) (1:1000) as an
16
internal control. The membrane was incubated with horseradish peroxidase-conjugated secondary
17
antibody (1:2000) for 1 h and detected using the Enhanced Chemiluminescence (ECL) Western blot
18
System (Amersham Biosciences).
19
Synthesis of miRNAs and sequences of miR-199a-5p inhibitors
20
MiR-199a-5p (sense: 5'-CCCAGUGUUCAGACUACCUGUUC-3' , antisense:
21
5'-ACAGGUAGUCUG AACACUGGGUU-3') and its antisense oligonucleotides (AMOs:
22
5'-GAACAGGTAGTCTGAACA CTGGG-3') were synthesized by Integrated DNA Technologies 7
Page 8 of 36
1
(IDT). Additionally, a scrambled RNA was used as negative control (NC), sense:
2
5'-UUCUCCGAACGUGUCACGUTT-3' and antisense: 5'-ACGUGACACGUUCGGAGAATT-3'.
3
DNA fragments of the 3'- UTRs of WNT2 and FZD4 mRNA containing the putative miR-199a-5p
4
binding sequence were synthesized by Invitrogen. These fragments were then respectively cloned
5
into the multiple cloning sites downstream the luciferase gene (HindIII and SpeI sites) in the
6
pMIR-REPORTTM luciferase miRNA expression reporter vector (Ambion, Inc.), as described
7
elsewhere(Yang, et al. 2007).
8
Transfection of miR-199a-5p/AMO-199a-5p in osteoblasts.
9
The MC3T3-E1 cells/primary osteoblasts were cultured with differentiation medium for 24 h.
10
Osteoblasts cultured in 6-well plates was divided into different groups. Osteoblasts in the control
11
group were cultured in differentiation culture medium. MicroRNA group were transfected with
12
miR-199a-5p or/and inhibitors for 48 h under differentiation condition, with X-tremeGENE siRNA
13
Transfection Reagent (Roche, Basel, Switzerland), as according to the manufacturer’s instructions.
14
Dexamethasone (Dex) (Sigma-Aldrich, St Louis, USA) group were treated with 10 -7M Dex for 5
15
days. MicroRNA together with Dex group cells were transfected with miR-199a-5p or/and inhibitors
16
for 48h under 10 -7M Dex and then changed with Dex for 3 days.
17
Luciferase Activity Assay
18
After 24 hours starvation in serum-free medium, HEK293 cells (1-105 per well) were transfected
19
with 1 µg miR-199a-5p/AMO-199a-5p or 1 µg PGL3-target DNA (firefly luciferase vector) and 0.1
20
µg PRL-TK (TK-driven Renilla luciferase expression vector), with Lipofectamine 2000 (Invitrogen),
21
as according to the manufacturer’s instructions. Luciferase activities were measured 48 hours after
22
transfection with a dual luciferase reporter assay kit (Promega) on a luminometer (Lumat LB9507). 8
Page 9 of 36
1
Quantification of miRNA levels
2
The mirVanaTM qRT-PCR miRNA Detection Kit (Ambion) was used in conjunction with real-time
3
PCR with SYBR Green I for quantification of miR-199a-5p transcript, as detailed elsewhere(Luo, et
4
al. 2007; Yang, et al. 2007).
5
Statistics
6
All data were analysed by using the SPSS 19.0 Software (SPSS, Inc.). The composite data are
7
expressed as means ± s.e.m. Statistical analysis was performed with one-way ANOVA followed by
8
Dunnett’s test where appropriate. Differences were considered to be significant at P≤0.05.
9
Results
10
The effect of Dex on differentiating osteoblasts proliferation.
11
To determine whether Dex regulated the proliferation of differentiating osteoblasts, we firstly used
12
CCK-8 assay to monitor cell proliferation. MC3T3-E1 cells under differentiation conditions were
13
treated with Dex at the concentrations (0, 10–9, 10–8, 10–7 M) from day 1 to day 5. Our results showed
14
that Dex decreased the proliferation of MC3T3-E1 cells in a dose- and time- dependent manner
15
(Figure 1A).
16
Furthermore, MC3T3-E1 cells were stimulated with 10–7 M Dex for 5 days prior to EdU assay.
17
EdU-stained photomicrographs and corresponding photomicrographs of total cells were shown in
18
Figure 1B. The proportion of cells with EdU-positive nuclei was shown in Figure 1C. EdU
19
incorporation was decreased in the Dex group, indicating that Dex could inhibit the MC3T3-E1 cells
20
proliferation.
21
To further examine proliferation, MC3T3-E1cells were cultured with 10–7 M Dex for 5 days and
22
stained with CFSE. CFSE irreversibly couples to cellular proteins. When cells divide, CFSE labeling 9
Page 10 of 36
1
is distributed equally between daughter cells, which are half as fluorescent as their parents. The peak
2
CFSE fluorescence intensity on flow cytometry was shifted by Dex, indicating that cells treated with
3
Dex had fewer cycles of cell replication as compared with the control group (Figure 1D).
4
Up-regulation of miR-199a-5p expression by Dex in differentiating osteoblasts.
5
To examine the role of miRNAs in the repression of osteoblasts proliferation by Dex, we firstly
6
detected some miRNAs expression, which have been reported to be related to bone formation and
7
mediate WNT signaling, in MC3T3-E1 cells exposed to 10–7 M Dex for 5 days. Several miRNAs
8
were found to be altered, in which miR-199a-5p was identified as a strong candidate responsible for
9
GC-decreased osteoblasts proliferation. We found that miR-574-3p and miR-27a were weakly
10
affected during this time frame. However, the expression of miR-199a-5p was significantly
11
upregulated by Dex (Figure 2A). Further studies demonstrated that Dex increased the expression of
12
miR-199a-5p in a time- dependent manner (Figure 2B). Similarly, the upregulation of miR-199a-5p
13
expression was also observed in primary osteoblasts treated with 10-7 M Dex for 5 days (Figure 2C).
14
Furthermore, we observed the effects of Dex on miR-199a-5p expression in vivo. We found that the
15
expression of miR-199a-5p was significantly upregulated in calvarias from mice treated with Dex
16
(Figure 2D). Thus, we hypothesized that miR-199a-5p might be involved in the process of
17
Dex-decreased osteoblasts proliferation.
18
Repression of differentiating osteoblasts proliferation by miR-199a-5p.
19
To delineate the role of miR-199a-5p in osteoblasts proliferation, we performed loss-of-function and
20
gain-of-function experiments in which we decreased and increased the quantities of miR-199a-5p
21
with miR-199a-5p inhibitor and miR-199a-5p mimic, respectively. MC3T3-E1 cells were transfected
22
with miR-199a-5p or/and AMO-199a-5p from day 1 to day 5. Then we assessed cell proliferation 10
Page 11 of 36
1
with CCK-8 assays. We found that overexpression of miR-199a-5p decreased MC3T3-E1 cells
2
proliferation. However, the depletion of miR-199a-5p with AMO-199a-5p resulted in the increased
3
MC3T3-E1 cells proliferation.
4
Furthermore, EdU assay by fluorescence microscope further demonstrated that miR-199a-5p mimic
5
clearly decreased MC3T3-E1 cells proliferation (Figure 3B,C), and miR-199a-5p inhibitor
6
significantly enhanced osteoblasts proliferation. Similar results were further confirmed by CFSE
7
fluorescence intensity assayment (Figure 3D). In addition, the effect of miR-199a-5p on osteoblasts
8
differentiation was observed by western blot. The expressions of osteogenic marker genes including
9
Runx2 and Osterix were significantly affected in MC3T3-E1 cells transfected with miR-199a-5p
10
or/and AMO-199a-5p for 48h, suggesting that miR-199a-5p was also involved in the process of
11
osteoblasts differentiation (Figure 3E).
12
Furthermore, primary osteoblasts were also used to test the effects of miR-199a-5p on cells
13
proliferation. The proliferation of primary osteoblasts transfected with miR-199a-5p or/and
14
AMO-199a-5p was measured by CCK8, EdU assay and CFSE fluorescence intensity assayment.
15
Similarly, we found that miR-199a-5p significantly decreased primary osteoblasts proliferation and
16
AMO-199a-5p enhanced it (Figure 4A-C).
17
could regulate osteoblasts proliferation. Involvement of miR-199a-5p in Dex-reduced osteoblasts
18
proliferation.
19
We consequently investigated whether miR-199a-5p was involved in Dex-decreased osteoblasts
20
proliferation. To this end, we observed the effect of Dex on proliferation in MC3T3-E1 cells
21
transfected with miR-199a-5p or/and AMO-199a-5p for 5 days. CCK-8 assay showed that
22
miR-199a-5p markedly enhanced Dex-decreased MC3T3-E1 cells proliferation. Furthermore, 11
Taken together, these results indicated that miR-199a-5p
Page 12 of 36
1
co-application of miR-199a-5p and AMO-199a-5p almost completely abolished the effect of
2
miR-199a-5p. Moreover, the inhibitory effect of Dex on MC3T3-E1 cells proliferation was
3
significantly alleviated when MC3T3-E1 cells were alone transfected with AMO-199a-5p, indicating
4
that miR-199a-5p was involved in Dex-decreased osteoblasts proliferation (Figure 5A). Similar
5
results were further confirmed by EdU assay and flow cytometric analysis of CFSE intensity (Figure
6
5B-C). All of above results were further verified in primary osteoblasts (Figure 6A-C).
7
Repression of WNT signaling by miR-199a-5p transfection.
8
Based on the above observations, miR-199a-5p was involved in osteoblasts proliferation. It is
9
possible that miR-199a-5p targets several regulatory factors related to osteoblasts proliferation. To
10
address this issue, we used a computation and bioinformatics-based approach to predict the putative
11
targets related to proliferation through TargetScan, which is hosted by the Wellcome Trust Sanger
12
Institute. These explorations lead to the identification of candidate targets of miR-199a-5p: WNT
13
signaling including FZD4 and WNT2 (Figure 7A,B). Western blot analysis of FZD4 and WNT2
14
expression in the MC3T3-E1 cells treated with 10-7 M Dex for 5 days are showed in Figure 7C,D.
15
We found that Dex decreased FZD4 and WNT2 expression in MC3T3-E1 cells. To prove that FZD4
16
and WNT2 are indeed repressed posttranscriptionally by miR-199a-5p, we determined the effect of
17
the miR-199a-5p on protein expression. Western blot analysis showed that miR-199a-5p lowered
18
markedly the levels of FZD4 and WNT2 proteins in MC3T3-E1 (Figure 7E,F). Co-application of
19
miR-199a-5p and AMO-199a-5p abolished almost completely the effect of miR-199a-5p. Moreover,
20
application of the AMO-199a-5p alone increased the levels of FZD4 and WNT2 in MC3T3-E1,
21
indicating that there is a basal level of miR-199a-5p activity in osteoblasts (Figure 7E,F). Similarly,
22
we also test the effects of miR-199a-5p on the levels of FZD4 and WNT2 proteins in primary 12
Page 13 of 36
1
osteoblasts. Our results showed that miR-199a-5p significantly decreased FZD4 and WNT2 proteins
2
expression and AMO-199a-5p increased it (Figure 8A,B). In addition, we observed the effect of
3
miR-199a-5p on the expression of B-catenin, a key mediater of WNT signaling pathway, in primary
4
osteoblasts. We found that miR-199a-5p decreased B-catenin expression, suggesting that
5
miR-199a-5p could inhibit WNT signaling pathway (Figure 8B).
6
Verification of interactions between miR-199a-5p and their target genes.
7
We constructed chimeric vectors by placing the 3'-UTRs of FZD4 and WNT2 into the 3'-UTR of a
8
luciferase reporter plasmid. We performed luciferase reporter assays in HEK293 cells that do not
9
express miR-199a-5p (Data not shown). Compared with the NC, transfection of miR-199a-5p with
10
the luciferase reporter gene linked to the 3'-UTR of FZD4 or WNT2 resulted in a significant decrease
11
of luciferase activity, and co-application of miR-199a-5p with AMO-199a-5p alleviated the decrease
12
of luciferase activity, whereas AMO-199a-5p alone had no effect (Figure 9A,B). These data suggest
13
that FZD4 and WNT2 are the targets of miR-199a-5p.
14
Successful delivery of miR-199a-5p, AMO-199a-5p and NC to the cells were further verified by
15
comparing the miR-199a-5p levels 48 hours after transfection of the constructs in MC3T3-E1 cells
16
and primary osteoblasts. Transfection resulted in significant increasing in miR-199a-5p levels
17
(Figure 9C). It is worth noting that the miR-199a-5P levels were dynamic after transfection. Our data
18
were collected at a specific time which was 48 hours after transfection, because the plateau level was
19
reached then. These results proved the feasibility of all experiments.
20
Discussion
21
Increased concentrations of GCs could cause the development of Cushing’s syndrome, with severe
22
osteoporosis. Previous studies have revealed that GCs have potent inhibitory effects on osteoblasts 13
Page 14 of 36
1
proliferation(Hong, et al. 2011; Walsh, et al. 2001). However, the molecular mechanisms involved in
2
the GC-inhibited osteoblasts proliferation are still poorly understood. In this study, we have
3
illustrated that miR-199a-5p plays a significant role in the process of GC-decreased osteoblasts
4
proliferation, by regulating WNT signaling pathway.
5
Several recent reports have suggested that GCs exert post-transcriptional control through the
6
regulation of miRNAs processing and expression(De Iudicibus, et al. 2013). Xing et al.reported that
7
GCs induced apoptosis by inhibiting miRNAs cluster miR-17~92 expression in chondrocytic
8
cells(Xing, et al. 2014). Furthermore, miR-29b and miR-29c were reported to be involved in
9
Toll-like receptor control of GC-induced apoptosis in human plasmacytoid dendritic cells(Hong, et al.
10
2013). In addition, miRNAs could also negatively regulate the GC receptor (GR) transcriptional
11
response by directly targeting the 3'-UTR of GRα mRNA(Lv, et al. 2012). It is also known that
12
osteoblasts are critical target cells of GCs. Our previous studies showed that miR-17~92a
13
downregulation by GCs leads to Bim targeting and induction of osteoblasts apoptosis(Guo, et al.
14
2013). Furthermore, GCs could also increase Receptor activator of nuclear factor B ligand (RANKL)
15
expression by down-regulation of miR-17/20a in osteoblasts, which indirectly enhances
16
osteoclastogenesis and bone resorption(Shi, et al. 2014). However, to our knowledge, there have
17
been no reports on whether miRNAs expression could be regulated by GCs in osteoblasts
18
proliferation. The present study is the first effort to observe the effect of GCs on miRNAs in
19
osteoblasts proliferation.
20
In this study, we detected some miRNAs expression in osteoblasts exposed to GCs. These miRNAs
21
have been reported to be related to cell proliferation and bone formation in previous
22
studies(Chiyomaru, et al. 2013; Wu, et al. 2013; Taipaleenmaki, et al. 2012; Huang, et al. 2012; Ma, 14
Page 15 of 36
1
et al. 2012; Wei, et al. 2012; Xu, et al. 2012). Furthermore, these miRNAs could mediate WNT
2
signaling pathway(Hashemi, et al. 2015; Guo, et al. 2014; Rathod, et al. 2014; Nagano, et al. 2013;
3
Liu, et al. 2013; Chiyomaru, et al. 2013), a curcial factor of GC-decreased bone formation. Therefore,
4
we firstly examined whether these six miRNAs were involved in the regulation of osteoblasts
5
proliferation by GC. Our results showed that only miR-199a-5p was identified as a strong candidate
6
responsible for GC-decreased osteoblasts proliferation. We speculated that miRNAs might play
7
different roles in different cells. Our study did not apply miRNAs chip to screen miRNAs expression
8
in response to GC-decreased osteoblasts proliferation. Therefore, several differentially expressed
9
miRNAs may be missed. Future studies are required to identify other miRNAs involved in
10
GC-inhibited osteoblasts proliferation.
11
Our results identified miR-199a-5p was significantly increased during Dex-decreased osteoblasts
12
proliferation. Furthermore, overexpression of miR-199a-5p decreased osteoblasts proliferation.
13
However, the depletion of miR-199a-5p with AMO-199a-5p resulted in the increased proliferation of
14
osteoblasts. Previous studies showed that miR-199a-5p was a ubiquitously expressed miRNA, whose
15
expression was modulated at key time points of development, growth, regeneration, cancers(Shi, et
16
al. 2014; Alexander, et al. 2013; Xu, et al. 2012). Shi et al. showed that miR-199a-5p affected
17
porcine preadipocyte proliferation and differentiation(Shi, et al. 2014). Alexander et al. found that
18
miR-199a-5p affected WNT signaling, cell proliferation, and myogenic differentiation(Alexander, et
19
al. 2013). Furthermore, Lentivirus-mediated overexpression of miR-199a-5p inhibited cell
20
proliferation of human hepatocellular carcinoma(Xu, et al. 2012). Although these studies have
21
reported the role of miR-199a-5p in the pathogenesis of several diseases, including breast cancer,
22
lymphoma or pulmonary hypertension, our study is the first report to uncover the role of 15
Page 16 of 36
1
miR-199a-5p in metabolic bone diseases and osteoblasts proliferation.
2
GCs, which regulate diverse physiological effects, have established both genomic and nongenomic
3
mechanisms(Stahn and Buttgereit 2008). The steroid’s nongenomic effects occur within seconds to
4
minutes and are mediated by the GR or by other means such as G protein-coupled receptors(Stahn
5
and Buttgereit 2008). It has been elucidated that GCs are unable to suppress bone formation in the
6
absence of GR expression in osteoblasts as their proliferation, differentiation, and apoptosis will
7
become immune to GCs(Rauch, et al. 2010). Therefore, GC-inhibited proliferation of osteoblasts is
8
triggered via GR-dependent transcriptional regulation. Previous study reported that GCs could
9
regulate miRNAs expression via a GR-mediated direct DNA binding mechanism(De Iudicibus, et al.
10
2013). In this study, we did not explore whether GR was involved in the regulation of miR-199a-5p
11
by GCs in osteoblasts. Therefore, further studies need to be done to uncover the progress of
12
GC-regulated miR-199a-5p in osteoblasts. WNT/ B-catenin signaling plays an important role in bone
13
development and metabolism by controlling both bone formation and resorption(Wang, et al. 2014).
14
Wnt ligands are secreted molecules that bind to cell surface receptors encoded by the Frizzled and
15
low-density lipoprotein receptor-related proteins (LRPs). Once bound, the ligands initiate a cascade
16
of intracellular events that activate the B-catenin activity and the DNA binding protein TCF, which
17
eventually result in the transcription of target genes(Wang, et al. 2014). It has been confirmed that
18
GCs could inhibit canonical WNT signaling to suppress bone formation(Guanabens, et al. 2014). In
19
this study, miR-199a-5p was involved in GC-inhibited osteoblasts proliferation through targeting
20
WNT signaling. Previous studies also showed that miR-199a-5p could target several cell
21
proliferation regulatory factors within the WNT signaling pathway, including FZD4 and
22
WNT2(Alexander, et al. 2013). 16
Page 17 of 36
1
Our data provide new evidence that miR-199a-5p plays a dominant effect in GC-inhibited
2
osteoblasts proliferation. The inhibitory effect of miR-199a-5p on osteoblasts proliferation is
3
ascribed to the disrupting of WNT signaling. This study is an effort to better understand the
4
molecular mechanism of GIO, and to provide new insights into the potential contribution of miRNAs
5
in the process of GC-mediated osteoblasts proliferation and bone formation.
6
Declaration of interest
7
The authors declare that there is no conflict of interest that could be perceived as prejudicing the
8
impartiality of the research reported.
9
Funding
10
This work was supported by the National Natural Science Foundation of China [Grant number.
11
81300713]; Innovation Program of Shanghai Municipal Education Commision [Grant number.
12
14YZ044]; and the Shanghai Jiaotong University School of Medicine Grant [Grant number.
13
13XJ10060].
14
Author contributions
15
L.G. and C.G.S. were involved in the conception and hypothesis delineation; L.G. and C.G.S.
16
designed the experiments, conducted the luciferase and quantitative real-time PCR experiments, and
17
wrote the article; J.Q., B.H., H.B.Z. and H.K. performed EdU staining and CFSE assay; P.H.,
18
modified the manuscript; M.J. and P.H. performed a part of the luciferase, western blot analysis;
19
L.F.D. designed and conducted the animal studies.
20
Acknowledgements
21
The authors thank Dr Zhaoping Qiu for expert technical assistance in carrying out Real time RT-PCR
22
and western blotting analysis. 17
Page 18 of 36
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Figure legends
2
Figure 1. The inhibition of differentiating osteoblasts proliferation by Dex. A: Proliferation of MC3T3-E1
3
cells was measured by CCK8 after cells were treated with 10–9, 10–8, 10–7 M Dex from day 1 to day 5. The
4
results showed that Dex decreased the viability of MC3T3-E1 cells in a dose- and time- dependent manner.
5
n = 3, **P < 0.01. B: Representative photomicrographs of EdU staining (top panel) and corresponding total
6
cell photomicrographs (middle panel). Blue: Hoechst labeling of cell nuclei; Red: EdU labeling of nuclei of
7
proliferative cells (×400). C: Quantitative data showing the percentage of EdU-positive cells in different
8
treatment groups (number of red vs number of blue nuclei). n = 3, **P < 0.01. D: MC3T3-E1 cells were
9
stained with CFSE before plating, cultured for 5 days, and analyzed by flow cytometry as described in
10
Materials and Methods. The peak CFSE fluorescence intensity on flow cytometry was shifted by Dex. n = 3.
11
Con indicates control, Dex indicates dexamethasone.
12
Figure 2. Up-regulation of miR-199a-5p expression by Dex in differentiating osteoblasts. A: qRT-PCR
13
analysis of miR-574-3p, miR-27a, miR-29a, miR-34a, miR-125b and miR-199a-5p expression in MC3T3-
14
E1 cells treated with 10-7 M Dex for 5 days. n = 3, **P < 0.01. B: qRT-PCR analysis of miR-199a-5p
15
expression in MC3T3-E1 cells treated with 10–7 M Dex from day 1 to day 5. n = 3, **P < 0.01. C: qRT-
16
PCR analysis of miR-199a-5p expression in primary osteoblasts treated with 10–7 M Dex for 5 days. n = 3,
17
**P < 0.01. D: qRT-PCR analysis of miR-199a-5p expression in the calvarias from mice treated with Dex
18
for 3 days. n = 3, **P < 0.01. Con indicates control, Dex indicates dexamethasone, miR indicates
19
microRNA.
20
Figure 3. Repression of MC3T3-E1 cells proliferation by miR-199a-5p. A: Proliferation of MC3T3-E1 cells
21
was measured by CCK8 after cells were transfected with miR-199a-5p or/and AMO-199a-5p from day 1 to
22
day 5. n = 3, **P < 0.01. B: Representative photomicrographs of EdU staining (top panel) and
23
corresponding total cell photomicrographs (middle panel). Blue: Hochest labeling of cell nuclei; Red: EdU 1
Page 25 of 36 1
labeling of nuclei of proliferative cells (×400). C: Quantitative data showing the percentage of EdU-positive
2
cells in different treatment groups (number of red vs number of blue nuclei). n = 3, **P < 0.01. D: MC3T3-
3
E1 cells transfected with miR-199a-5p or/and AMO-199a-5p were stained with CFSE and analyzed by flow
4
cytometry as described in Materials and Methods. n = 3. E: Western blot analysis of Runx2 and Osterix
5
expression in MC3T3-E1 cells transfected with miR-199a-5p or/and AMO-199a-5p. n = 3, **P < 0.01, *P
6
< 0.05. Con indicates control, NC indicates negative control, miR indicates microRNA.
7
Figure 4. Repression of primary osteoblasts proliferation by miR-199a-5p. A: Proliferation of primary
8
osteoblasts was measured by CCK8 after cells were transfected with miR-199a-5p or/and AMO-199a-5p
9
from day 1 to day 5. n = 3, **P < 0.01. B: Quantitative data showing the percentage of EdU-positive cells in
10
different treatment groups (number of red vs number of blue nuclei). n = 3, **P < 0.01, *P < 0.05. C:
11
Primary osteoblasts transfected with miR-199a-5p or/and AMO-199a-5p were stained with CFSE and
12
analyzed by flow cytometry as described in Materials and Methods. n = 3. Con indicates control, NC
13
indicates negative control, miR indicates microRNA.
14
Figure 5. Involvement of miR-199a-5p in Dex-reduced MC3T3-E1 cells proliferation. A: Proliferation of
15
MC3T3-E1 cells was measured by CCK8 after cells were transfected with miR-199a-5p or/and AMO-199a-
16
5p under Dex from day 1 to day 5. n = 3, **P < 0.01. B: Quantitative data showing the percentage of EdU-
17
positive cells in different treatment groups (number of red vs number of blue nuclei). n = 3, **P < 0.01. C:
18
MC3T3-E1 cells transfected with miR-199a-5p or/and AMO-199a-5p under Dex were stained with CFSE
19
and analyzed by flow cytometry as described in Materials and Methods. n = 3. Con indicates control, Dex
20
indicates dexamethasone, NC indicates negative control, miR indicates microRNA.
21
Figure 6. Involvement of miR-199a-5p in Dex-reduced primary osteoblasts proliferation. A: Proliferation of
22
primary osteoblasts was measured by CCK8 after cells were transfected with miR-199a-5p or/and AMO-
23
199a-5p under Dex from day 1 to day 5. n = 3, **P < 0.01. B: Quantitative data showing the percentage of 2
Page 26 of 36 1
EdU-positive cells in different treatment groups (number of red vs number of blue nuclei). n = 3, **P
