Background/Aims: Osteoporosis is a progressive bone disease characterized by a decrease in bone mass and density, which results in an increased risk of fractures. Mesenchymal stem cells (MSCs) are progenitor cells that can differentiate into osteoblasts, osteocytes and adipocytes in bone and fat formation. A reduction in the differentiation of MSCs into osteoblasts contributes to the impaired bone formation observed in osteoporosis. MicroRNAs (miRNAs) play a regulatory role in osteogenesis and MSC differentiation. MiR-27a has been reported to be down-regulated in the development of osteoporosis and during adipogenic differentiation. Methods: In this study, a miRNA microarray analysis was used to investigate expression profiles of miRNA in the serum of osteoporotic patients and healthy controls and this data was validated by quantitative real-time PCR (qRT-PCR). MSCs isolated from human and mice with miR-27a inhibition or overexpression were induced to differentiate into osteoblasts or adipocytes. TargetScan and PicTar were used to predict the target gene of miR-27a. The mRNA or protein levels of several specific proteins in MSCs were detected using qRT-PCR or western blot analysis. Ovariectomized mice were used as in vivo model of human postmenopausal osteoporosis for bone mineral density measurement, micro-CT analysis and histomorphometric analysis. Results: Here, we analyzed the role of miR-27a in bone metabolism. Microarray analysis indicated that miR-27a expression was significantly reduced in osteoporotic patients. Analysis on MSCs derived from patients with osteoporosis indicated that osteoblastogenesis was reduced, whereas adipogenesis was increased. MSCs that had undergone osteoblast induction showed a significant increase in miR-27a expression, whereas cells that had undergone adipocyte induction showed a significant decrease in miR-27a expression, indicating that miR-27a was essential for MSC differentiation. We demonstrated that myocyte enhancer factor 2 c (Mef2c), a transcription factor, was the direct target of miR-27a using a dual luciferase assay. An inverse relationship between miR-27a expression and Mef2c expression in osteoporotic patients was shown. Silencing of miR-27a decreased bone formation, confirming the role of miR-27a in bone formation in vivo. Conclusion: In summary, miR-27a was essential for the shift of MSCs from osteogenic differentiation to adipogenic differentiation in osteoporosis by targeting Mef2c.

Osteoporosis is a bone metabolic disease characterized by a systemic impairment of bone mass and increase in bone marrow (BM) fat, which results in increased propensity of fragility fractures. Mesenchymal stem cells (MSCs) can undergo multilineage differentiation into a variety of connective tissue cell types. For example, they are the progenitor cells of osteoblasts, osteocytes and adipocytes in bone and fat formation [1,2]. A reduction in the differentiation of MSCs into osteoblasts contributes to the impaired bone formation observed in osteoporosis. It has been reported that adipose tissue in BM is inversely related to bone formation in osteoporosis, and patients with a high bone mass phenotype show inhibition of adipogenesis [3,4]. It had been suggested that down-regulation of hormones, cytokines or transcription factors could result in the MSCs lineage commitment switch.

Recently, emerging evidence show that microRNAs (miRNAs) are crucial for physiological bone development and MSCs differentiation. MiRNAs are small endogenous non-coding RNAs (18-25 nucleotides in length) that negatively regulate gene expression through incomplete base-pairing to the 3ʹ untranslated region (3ʹ-UTR) of target mRNAs [5,6,7,8,9]. Increasing evidence suggests that miRNAs play a role in the regulation of diverse biological and pathological processes, such as developmental timeline, organogenesis, apoptosis, cell proliferation and differentiation [10,11]. Several miRNAs have been reported to play a role in bone formation, such as miR-20a, miR-26, miR-27, miR-29 family, miR-30 family, miR-15 family and miR-206 [12,13,14,15,16,17,18,19,20,21]. In our previous study, we found the expression of many miRNAs was significantly changed in the serum of osteoporotic patients compared with the healthy controls. In particular, miR-27 appears from the evidence to play an important role in adipogenic and osteogenic differentiation. Wang et al. reported that miR-27 promoted osteoblast differentiation through activating Wnt signaling, implying that miR-27 might be down-regulated in the development of osteoporosis [22]. Pan et al. showed that miR-27a promoted the development of osteosarcoma by targeting MAP2K4 via JNK/p38 signaling pathway [23]. Interestingly, Lin et al. demonstrated that the miR-27 family was also down-regulated during adipogenic differentiation [24]. It is known that the balance between adipogenic and osteogenic differentiation is closely correlated with osteoporosis, the present study was therefore undertaken to investigate the role of miR-27a in bone metabolism, with the aim of gaining insight into the pathological mechanism of osteoporosis.

Study participants

The research described here complied with the World Medical Association Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects. This study was approved by the Institutional Ethical Committee and written informed consent was obtained from patients before samples were taken. There were a total of 155 participants in the study, including 81 women with postmenopausal osteoporosis (51-62 years old) and 74 healthy premenopausal women (40 - 46 years old) from Shanghai First People's Hospital between April 2012 and June 2014. The inclusion criteria were as follows: natural menopause after 40 years of age and a bone mineral density (BMD) of at least 1.0 SD below the peak mean bone density of healthy young women (−1.0 T-score) at the posterior-anterior lumbar spine 1-4 (L1-L4), total hip or femoral neck. Subjects with a medical history of osteoporosis treatment, hormone replacement therapy, early menopause (age < 40), abnormal menopause, acute inflammation of the gastrointestinal tract, or chronic renal failure were excluded.

BMD measurement

BMD was evaluated by a dual-energy X-ray absorptiometry (DXA; GE Healthcare, Madison, Wisconsin, USA) at the lumbar spine, total hip and femoral neck. The DXA instruments were calibrated and reference values were obtained as previously described [25]. Regions of severe scoliosis, vertebral fracture, and operated on sites were excluded from BMD measurements.

miRNA microarray

A miRNA microarray platform was used to determine the expression profiles of miRNA in 5 women with postmenopausal osteoporosis and 5 healthy premenopausal women. The array was conducted using the commercially available G4471A Human miRNA Microarray (Agilent Technologies, Santa Clara, CA, USA), which consists of 961 probes for 851 human miRNAs, based on Sanger miRBase release 12.0. The arrays were washed and scanned with a laser confocal scanner (G2565BA; Agilent Technologies) according to the manufacturer's instructions. The intensities of fluorescence were calculated by Feature extraction software (Agilent Technologies). Differentially expressed miRNAs were identified by arbitrarily setting the threshold at a fold change of 2.0 or above combined with p < 0.05 (ANOVA).

MSC isolation

Human MSCs were isolated and expanded from aspirates of BM from informed different donors. In brief, mononuclear cells were isolated using Ficoll-Hypaque density gradient centrifugation and plated at 1 × 106/mL in DMEM-low glucose supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), and antibiotics, 2 mM glutamine (all from Invitrogen, Carlsbad, CA, USA). Medium was replaced and non-adherent cells were removed after 2 days of initial culture and every 3 or 4 days thereafter. When cells reached confluent, adherent cells were detached with trypsin/ethylenediaminetetraacetic acid (EDTA) (all from Sigma-Aldrich, Munich, Germany) and reseeded for expansion. Cells after three passages were used in the experiments.

Mouse MSCs were isolated from the BM of tibias and femora of 6- to 10-wk-old BALB/c female mice. Animal protocols were approved by the Institutional Review Board of the Institute of Health Sciences. Cells were cultured in DMEM medium supplemented with 10% FBS, 2 mM glutamine, and antibiotics (all from Invitrogen). Non-adherent cells were removed after 24 h, and adherent cells were maintained with medium that was replenished every 3 d. To obtain MSC clones, cells at confluence were harvested and seeded into 96-well plates by limited dilution. Individual clones were then picked and expanded. Cells were used at the 5th to 15th passage.

MSC differentiation assay

MSCs at passage 8 were induced towards the osteogenic and adipogenic lineages. For osteogenic induction, MSCs were seeded in bone induction medium with LGDMEM (Gibco-BRL, Gaithersburg, MD, USA) containing 10% FBS, penicillin (10 kU/ml), streptomycin (10 mg/ml), glutamine (2 mM), supplemented with dexamethasone (10 nM), L-ascorbic acid-2-phosphate (0.1 mM), beta-glycerol phosphate (5 mM) (all from Sigma) and BMP-2 (100 ng/ml) (Tebu-Bio, Magenta, Italy). After 2 wk, differentiated MSCs and control cells were stained with aqueous 0.5% (v/v) Alizarin Red-S (Sigma) and washed with PBS with 10% (v/v) cetylpiridinium (Sigma). For adipogenic induction, MSCs were plated in DMEM supplemented with 10% rabbit serum (Euroclone, Paignton, UK), 5% horse serum (Hyclone), 1 mM dexamethasone, 60 nM indomethacin, 10 mM rh-insulin and 0.5 mM isobuthylmethylxanthine. After 10 d, differentiation was verified by Oil-Red-O (Sigma) staining.

Cell transfection and plasmid construction

For the MSC transfection assay, the plasmid or oligonucleotide was transfected into cells using Lipofectamine RNAiMAX kit (Invitrogen) at ∼50% confluence, following the manufacturer's instructions.

The human myocyte enhancer factor 2 c (Mef2c) coding sequence was cloned into lentivirus pCDH vector to generate stably transfected cell lines. Viral production was performed in 293T cells. 293T cells were cotransfected with lentiviral vector pCDH and packaging plasmid. Cells were incubated overnight at 37°C and 5% CO2. The supernatant was collected at 24 and 48 h post-transfection. A fragment of the Mef2c 3′UTR containing the predicted binding site for miR-27a and the binding site mutated (mut) 3′UTR were inserted into psiCHECK2 vector for the dual luciferase reporter assay.

Inhibition or overexpression of miR-27a in MSCs

Antisense oligonucleotide inhibitor of miR-27a (anti-miR-27a) was used to suppress the function of miR-27a in MSCs from healthy donor. Briefly, anti-miR-27a and control oligonucleotide were transfected into MSCs using Lipofectamine RNAiMAX kit (Invitrogen) at ∼50% confluence, following the manufacturer's instructions. And synthetic miR-27a mimic was ued to overexpress miR-27a in MSCs from osteoporotic patient, by transfecting the mimic into MSCs as described obove. The sequence of miR-27a mimic was as follows: uucacaguggcuaaguuccgc.

Gene silencing using shRNA

In the gene knockdown assay, Mef2c specific shRNA, and scrambled shRNA were prepared to transfect cells. The shRNA was mixed with Lipofectamine™ 2000 (Invitrogen) and transfected into cells. Then cells were cultured and incubated at 37°C for 6 h, followed by incubation with complete medium for additional 24 h. Then cells were harvested for further study.

Selected target mRNAs for miR-27a

Target mRNAs for miR-27a were predicted using TargetScan (www.targetscan.org/), PicTar (http://pictar.mdc-berlin.de/). Gene Ontology was used in a subsequent screening step.

Dual-luciferase reporter assays

Luciferase reporter assays were carried out using the Dual-Luciferase Reporter Assay System (psiCHECK-2 vector; Promega, Madison, WI, USA). A fragment of the Mef2c 3′UTR containing the predicted binding site for miR-27a and the binding site mut 3′UTR were inserted into psiCHECK2 vector. All constructs were verified by DNA sequencing. The psiCHECK-2 vector containing wild-type (wt) or mut Mef2c was transfected into cells with or without the synthetic miR-27a mimic. Thirty-six hours after transfection, luciferase activity was detected using a dual-luciferase reporter assay system and normalized to Renilla activity.

Quantitative real-time PCR (qRT-PCR)

The serum miRNAs were enriched using the mirVana microRNA Isolation kit (Applied Biosystems, Foster City, CA, USA) after total RNA was isolated by TRIzol Reagent (Invitrogen). The expression of miR-27a was determined by Taqman microRNA assay kit especially for miR-27a (Applied Biosystems) according to manufacturer's instructions. For data analysis, we used the U6 RNA as an endogenous control. Total RNA of MSCs was also extracted using TRIzol Reagent. Expression of alkaline phosphatase (ALP), RUNX2, osteocalcin (OCN), Peroxisome proliferator-activated receptor (PPAR)-γ, and lipoprotein lipase (LPL) was measured by qRT-PCR. Quantitative RT-PCR reactions were performed using the SYBR Green PCR mix on an ABI Prism 7900HTthermocycler. Thermocyclerconditions included 2-minute incubation at 50°C, then 95°C for 10 minutes; this was followed by a 2-step PCR program, as follows: 95°C for15 seconds and 60°C for 60 seconds for 40 cycles. β-Actin was used as an internal control to normalize for differences in the amount of total RNA in each sample. Fold changes were calculated using the 2−ΔΔCt method. All procedures were repeated triply. Primers are listed in Table 1.

Table 1

qRT-PCR primers

qRT-PCR primers
qRT-PCR primers

Western blot analysis

Cells or bones were lysed with RIPA containing protease and phosphatase inhibitors (Roche Applied Science, Mannheim, Germany). Equal amounts of protein were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), immunoblotted with primary Ab (Mef2c and β-actin (Abcam, Cambridge, MA, USA), 1:1000 dilution), and visualized with horseradish-peroxidase-coupled secondary Ab.

Animal model

Sixty 8-week-old BALB/c female mice weighing 19-21 g were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). There was no significant difference in the initial body weights of the mice. The mice were housed in standard cages (4 mice per cage) and maintained at 22 ± 5°C with constant humidity (50 ± 10%) and a 12 h light-dark cycle. These animals had free access to autoclaved water and a pellet diet for 1 wk prior to the surgery. Next, a sham-operation was performed on the mice or the mice were surgically ovariectomized (OVX) after anaesthesia by pentobarbital sodium (50 mg/kg body weight, i.p.).The ovariectomy operation was performed according to Steven K. Boyd's procedure [26]. The mice were randomly divided into 6 groups (n = 10 per group): group 1, sham mice with PBS treatment; group 2, sham mice with mut antagomiR-27a treatment; group 3, sham mice with antagomiR-27a treatment; group 4, OVX mice with PBS treatment; group 5, OVX mice with mut antagomiR-27a treatment; group 6, OVX mice with antagomiR-27a treatment.

Treatment of mice with antagomiR-27a, BMD measurement, and micro-CT analysis

Mice were injected i.v. with PBS, mut antagomiR-27a, or antagomiR-27a (three injections of 80 mg/kg/d in the first wk, and another injection on d 1-3 of the fourth wk) and bones were harvested 6 wk after the first injection. The left femur of each mouse was fixed onto the scanning table along the longitudinal axis, and the whole femur was scanned by DXA using a PIXImus densitometer (GE Healthcare) to determine the BMD. The femur of each mouse was then fixed with 4% paraformaldehyde for 24 h and subsequently washed with 10% saccharose solution; 12 h later, structure parameter bone volume/tissue volume ratio (BV/TV) was measured by micro-CT scanning using the GE explore Locus SP system (GE Healthcare).

Histomorphometric analysis

For histological analyses, the mice were injected with 25 mg/kg calcein at 8 and 2 d before euthanasia. The femurs were fixed in 70% ethanol, dehydrated in increasing concentrations of ethanol, and the undecalcified bones were embedded in methyl methacrylate. Serial 5-µm sections in the proximal region of the femur were made using a microtome. The parameters obtained for the bone formation were bone formation rate over bone surface area (BFR/BS) and osteoblast surface area over bone surface area (Ob.S/BS). The parameters measured for bone resorption were osteoclast surface area over bone surface area (Oc.S/BS) and the number of osteoclasts per bone perimeter (N.Oc/B.Pm).

Statistical analysis

SPSS 15.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All quantitative data were expressed as the mean ± S.E.M. of at least three separate experiments, using a two-tail Student's test to compare two independent groups.

MiR-27a is significantly down-regulated in postmenopausal osteoporotic patients

The clinical characteristics of the participants are presented in Table 2. Participants with osteoporosis were significantly older than those without. The mean values for height, weight, and BMI were lower in osteoporotic patients. Besides, BMD at all measured sites was also significantly lower in osteoporotic patients, as expected.

Table 2

Clinical characteristics of participants. Abbreviations: BMI, body mass index; BMD, bone mineral density

Clinical characteristics of participants. Abbreviations: BMI, body mass index; BMD, bone mineral density
Clinical characteristics of participants. Abbreviations: BMI, body mass index; BMD, bone mineral density

We used a miRNA microarray platform which covers a total of 851 human miRNAs to investigate expression profiles of miRNA in the serum of five women with postmenopausal osteoporosis versus five healthy premenopausal women. The median values of four background corrected replicas for each miRNA capture probe were uploaded into the microarray analysis software for comparison. As listed in Table 3, 33 miRNAs were down-regulated >2-fold in the serum of osteoporotic patients as compared with the healthy controls. MiR-27a was one of the most strongly down-regulated miRNAs in the serum of osteoporotic patients, which was expected, as it had already been shown essential in adipogenic and osteogenic differentiation. To validate the microarray data, we further examined the expression of miR-27a in the serum of 81 osteoporotic patients and 74 healthy controls. Consistent with the data obtained from microarray, the average expression level of miR-27a was significantly lower in the serum of osteoporotic patients than that of healthy controls (Fig. 1A). The expression of miR-27a decreased by 2-fold or higher in 77 of 81 osteoporotic patients (95.06%) and lower than 2-fold in the remaining four samples (4.94%) when compared with healthy controls (Fig. 1B). Therefore, miR-27a was selected for further study.

Table 3

Downregulated miRNAs in the serum of osteoporosis patients

Downregulated miRNAs in the serum of osteoporosis patients
Downregulated miRNAs in the serum of osteoporosis patients
Fig. 1

miR-27a is significantly down-regulated in postmenopausal women with osteoporosis. (A) miR-27a expression was examined by qRT-PCR in the serum of osteoporotic patients (n = 81) and healthy controls (n = 74). Data were normalized to U6 RNA. (B) miR-27a expression was frequently decreased more than or equal to 2-fold in the serum of osteoporotic patients (95.06%), as shown in the clustering diagram. *, p < 0.05.

Fig. 1

miR-27a is significantly down-regulated in postmenopausal women with osteoporosis. (A) miR-27a expression was examined by qRT-PCR in the serum of osteoporotic patients (n = 81) and healthy controls (n = 74). Data were normalized to U6 RNA. (B) miR-27a expression was frequently decreased more than or equal to 2-fold in the serum of osteoporotic patients (95.06%), as shown in the clustering diagram. *, p < 0.05.

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MSCs from osteoporotic patients shift the differentiation ability from osteoblasts to adipocytes

To analyze the differentiation ability of MSCs in osteoporotic patients, MSCs isolated from osteoporotic patients and the healthy controls were induced to differentiate into osteoblasts or adipocytes. Alizarin Red-S and Oil-Red-O were used to stain osteoblasts and adipocytes respectively. As shown in Fig. 2A, osteoblastogenesis was reduced in MSCs obtained from osteoporotic patient as compared with that from the healthy controls, whereas adipogenesis was increased in osteoporotic patients compared with that of the healthy controls. To confirm these observations, the expression of specific markers for osteoblasts (ALP, RUNX2 and OCN) and adipocytes (PPARγ and LPL) were measured by qRT-PCR. The osteoblast specific markers showed significantly reduced expression in MSCs from osteoporotic patient as compared with MSCs from the healthy controls (p < 0.05) (Fig. 2B), whereas the adipocyte specific markers showed significantly increased expression in osteoporotic patients compared with the healthy controls (p < 0.05) (Fig. 2C). These results confirmed our microscopic observations.

Fig. 2

Osteoporotic patient-derived MSCs shift the differentiation ability from osteoblasts to adipocytes. (A-C) MSCs at passage 8 were isolated from osteoporotic patients (n = 3) and healthy controls (n = 3) were induced to differentiate into osteoblasts or adipocytes in differential medium. Osteoblast differentiation indicated by Alizarin Red-S staining at week 2 and adipocyte differentiation indicated by Oil-Red-O staining at day 10 were shown. Scale bar, 500 µm (A). Expression of specific markers for osteoblasts (ALP, RUNX2 and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR (n = 3). Data are normalized to expression level of β-actin (B, C). Results are mean ± S.E.M. from three independent experiments. (D-F) MSCs isolated from sham and ovariectomy (OVX) mice were induced to differentiate into osteoblasts or adipocytes. Osteoblast differentiation indicated by Alizarin Red-S staining at week 2 and adipocyte differentiation indicated by Oil-Red-O staining at day 10 were shown. Scale bar, 500 µm (D). Expression of specific markers for osteoblasts (ALP, RUNX2 and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR (n = 3). Data are normalized to expression level of β-actin (E, F). Results are mean ± S.E.M. from three independent experiments. *, p < 0.05.

Fig. 2

Osteoporotic patient-derived MSCs shift the differentiation ability from osteoblasts to adipocytes. (A-C) MSCs at passage 8 were isolated from osteoporotic patients (n = 3) and healthy controls (n = 3) were induced to differentiate into osteoblasts or adipocytes in differential medium. Osteoblast differentiation indicated by Alizarin Red-S staining at week 2 and adipocyte differentiation indicated by Oil-Red-O staining at day 10 were shown. Scale bar, 500 µm (A). Expression of specific markers for osteoblasts (ALP, RUNX2 and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR (n = 3). Data are normalized to expression level of β-actin (B, C). Results are mean ± S.E.M. from three independent experiments. (D-F) MSCs isolated from sham and ovariectomy (OVX) mice were induced to differentiate into osteoblasts or adipocytes. Osteoblast differentiation indicated by Alizarin Red-S staining at week 2 and adipocyte differentiation indicated by Oil-Red-O staining at day 10 were shown. Scale bar, 500 µm (D). Expression of specific markers for osteoblasts (ALP, RUNX2 and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR (n = 3). Data are normalized to expression level of β-actin (E, F). Results are mean ± S.E.M. from three independent experiments. *, p < 0.05.

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To confirm our findings obtained from osteoporotic patients, we analyzed the differentiation ability of MSCs isolated from ovariectomy (OVX) mice or healthy control mice. MSCs from mice were induced to differentiate into osteoblasts or adipocytes and were then stained with Alizarin Red-S or Oil-Red-O, respectively. Similar to our findings in MSCs from osteoporotic patients, in the MSCs of OVX mice osteoblastogenesis was reduced compared with the cells of the control mice, whereas adipogenesis was increased in OVX mice compared with the control mice (Fig. 2D). Furthermore, osteoblast specific markers (ALP, RUNX2 and OCN) showed significantly reduced expression in the MSCs of OVX mice compared with cells from the control mice (p < 0.05) (Fig. 2E), whereas adipocyte specific markers showed significantly increased expression in OVX mice compared with the control mice (p < 0.05) (Fig. 2F) by qRT-PCR. These results confirmed our findings from osteoporotic patients that osteoblastogenesis was down-regulated whereas adipogenesis was up-regulated.

MiR-27a is essential in MSC differentiation

MSCs, isolated from the healthy human controls, were induced to differentiate into osteoblasts or adipocytes and expression of miR-27a was measured up to 3 wk following induction. Cells that had undergone osteoblast induction showed a significant increase in expression levels of miR-27a from 0-3 wk (Fig. 3A). Cells that had undergone adipocyte induction showed a significant decrease in expression levels of miR-27a from 0-2 wk, with expression levels reaching 0 at 2 wk following induction (Fig. 3B).

Fig. 3

miR-27a is essential in MSC differentiation. MSCs at passage 8 were isolated from osteoporotic patients (n = 3) and healthy controls (n = 3) were induced to differentiate into osteoblasts or adipocytes in differential medium. (A, B) Expression of miR-27a during osteoblast and adipocyte induction of MSCs from the healthy controls (n = 3). Data were normalized to U6 RNA at each time point. Expression of miR-27a at 0 week were normalized to 1. (C, D) Anti-miR-27a oligonucleotide was used to inhibit miR-27a activity in MSCs. Expression of specific markers for osteoblasts (ALP, RUNX2and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR in the normal MSCs and anti-miR-27a MSCs (n = 3). (E, F) miR-27a mimic was transfected into MSC to overexpress miR-27a. Expression of specific markers for osteoblasts (ALP, RUNX2 and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR in osteoporotic patient-derived MSCs with or without miR-27a overexpression (n = 3). Results are mean ± S.E.M. from three independent experiments. *, p < 0.05.

Fig. 3

miR-27a is essential in MSC differentiation. MSCs at passage 8 were isolated from osteoporotic patients (n = 3) and healthy controls (n = 3) were induced to differentiate into osteoblasts or adipocytes in differential medium. (A, B) Expression of miR-27a during osteoblast and adipocyte induction of MSCs from the healthy controls (n = 3). Data were normalized to U6 RNA at each time point. Expression of miR-27a at 0 week were normalized to 1. (C, D) Anti-miR-27a oligonucleotide was used to inhibit miR-27a activity in MSCs. Expression of specific markers for osteoblasts (ALP, RUNX2and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR in the normal MSCs and anti-miR-27a MSCs (n = 3). (E, F) miR-27a mimic was transfected into MSC to overexpress miR-27a. Expression of specific markers for osteoblasts (ALP, RUNX2 and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR in osteoporotic patient-derived MSCs with or without miR-27a overexpression (n = 3). Results are mean ± S.E.M. from three independent experiments. *, p < 0.05.

Close modal

Next, the expression of specific markers for osteoblasts (ALP, RUNX2 and OCN) or adipocytes (PPARγ and LPL) was measured by qRT-PCR in normal MSCs and MSCs transfected with miR-27a inhibitor anti-miR-27a. Osteoblast specific markers showed significantly reduced expression in anti-miR-27a-transfected MSCs compared with normal MSCs (p<0.05) (Fig. 3C), whereas adipocyte specific markers showed significantly increased expression in anti-miR-27a-transfected MSCs compared with the normal MSCs (p < 0.05) (Fig. 3D) by qRT-PCR. The expression of these specific markers was then measured by qRT-PCR in MSCs from osteoporotic patients and MSCs in which miR-27a was overexpressed. Osteoblast specific markers showed significantly increased expression in miR-27a-overexpressed MSCs compared with MSCs from the osteoporotic patients (p < 0.05) (Fig. 3E), whereas adipocyte specific markers showed significantly decreased expression in miR-27a-overexpressed MSCs compared with MSCs from osteoporotic patients (p < 0.05) (Fig. 3F).

Mef2c is the direct target of miR-27a

MiRNAs negatively regulate gene expression through incomplete base-pairing to the 3ʹ-UTR of target mRNAs. Figure 4A shows the region of complementarity between the miR-27a sequence and the mef2c 3'UTR sequence, a putative target gene of miR-27a. In this study, we generated constructs comprising a fragment of the Mef2c 3′UTR containing the predicted binding site for miR-27a, and a mut version of the binding site containing a mutation in the 3′UTR. These constructs were inserted into psiCHECK2 vector and verified by DNA sequencing. A schematic representation of the Mef2c wt and mut 3´-UTR constructs was shown in Fig. 4A.

Fig. 4

Mef2c is the direct target of miR-27a. (A) The wild-type (wt) or mutant (mut) 3´-UTR of Mef2c was transfected into MSCs from healthy people. (B) Luciferase activity was determined 36 h after transfection (n = 3). Data were normalized to the luciferase activity of the transfected miR-control. (C, D) Expression of Mef2c after miR-27a overexpression at the protein level and the mRNA level in MSCs from healthy people were detected by western blot and qRT-PCR (n = 3). Data were normalized to the control. (E) The relationship of relative miR-27a and Mef2c expression in postmenopausal women with osteoporosis was shown. We have drawn the correlation formula: y=-0.2213x+1.8664 (x: expression level of miR-27a, y: expression level of Mef2c) (n = 26). All data were presented as mean ± S.E.M. from three independent experiments. *, p < 0.05.

Fig. 4

Mef2c is the direct target of miR-27a. (A) The wild-type (wt) or mutant (mut) 3´-UTR of Mef2c was transfected into MSCs from healthy people. (B) Luciferase activity was determined 36 h after transfection (n = 3). Data were normalized to the luciferase activity of the transfected miR-control. (C, D) Expression of Mef2c after miR-27a overexpression at the protein level and the mRNA level in MSCs from healthy people were detected by western blot and qRT-PCR (n = 3). Data were normalized to the control. (E) The relationship of relative miR-27a and Mef2c expression in postmenopausal women with osteoporosis was shown. We have drawn the correlation formula: y=-0.2213x+1.8664 (x: expression level of miR-27a, y: expression level of Mef2c) (n = 26). All data were presented as mean ± S.E.M. from three independent experiments. *, p < 0.05.

Close modal

The Mef2c wt and mut constructs were used in a dual luciferase reporter assay. The constructs were inserted into psiCHECK2 vector and the wt and mut vectors were transfected into cells with or without the synthetic miR-27a mimic. Luciferase activity was determined 36 h after transfection. Data were normalized to the luciferase activity of the transfected miR-control. In the presence of the miR-27a mimic, the luciferase activity of Mef2c wt was decreased, while miR-27a mimic has little effect on luciferase activity of Mef2c mut (Fig. 4B).

Next, the expression levels of Mef2c after miR-27a overexpression were analyzed at the protein level by western blot (Fig. 4C) and at the mRNA level by RT-PCR (Fig. 4D). A reduction in Mef2c expression levels were seen in the miR-27a-overexpressed cells compared with the miR control at the protein levels. However, no difference was observed in the mRNA level. Finally, the relationship between miR-27a and Mef2c protein levels in postmenopausal osteoporotic patients was analyzed and the values were plotted on the graph shown in Fig. 4E. An inverse relationship between miR-27a expression and Mef2c protein expression was observed.

MiR-27a regulates bone formation in vivo

Antagomirs, also known as anti-miRNAs, are synthetic oligonucleotides that prevent other molecules from binding to a target site on an mRNA molecule. It has previously been reported that mice treated with antagomiR-27a (that prevents miR-27a binding) have decreased bone mass and reduced osteoblast activity. In this study, to address the in vivo role of miR-27a in bone formation, mice were injected i.v. with PBS, mut antagomiR-27a or antagomiR-27a (three injections of 80 mg/kg/d in the first wk, and another injection on d 1-3 of the fourth wk) and bones were harvested 6 wk after the first injection.

Silencing of miR-27a by treatment of mice with antagomiR-27a resulted in upregulated expression of Mef2c as compared with PBS or mut antagomiR-27a controls between the PBS or mut antagomiR-27a controls and antagomiR-27a-treated OVX mice or control mice (Fig. 5A). In addition, a statistically significant decrease in BMD was also detected (p < 0.05) (Fig. 5B). A statistically significant decrease in bone volume/total volume ratio (BV/TV) was detected between the PBS or mut antagomiR-27a controls and antagomiR-27a-treated OVX mice or control mice (p < 0.05) (Fig. 5C). Furthermore, a statistically significant decrease in BMD and BV/TV was detected between the control mice and OVX mice (p < 0.05) (Fig. 5B and C).

Fig. 5

AntagomiR-27a-treated mice have decreased bone mass and reduced osteoblast activity. Sham or OVX mice were injected i.v. with PBS, mut antagomiR-27a, or antagomiR-27a (three injections of 80 mg/kg/d in the first wk, and another injection on d 1-3 of the fourth wk) and bones were harvested at 6 wk after the first injection (n = 60 for each group). (A) Expression of Mef2c was measured by western blot analysis. (B) BMD of femurs was measured using a PIXImus densitometer. (C) Structure parameter bone volume/tissue volume ratio (BV/TV) was measured by micro-CT. (D) Bone formation parameters of mouse femur, bone formation rate over bone surface area (BFR/BS) and osteoblast surface area over bone surface area (Ob.S/BS) were measured by histomorphometric analysis. (E) Bone resorption parameters of mouse femur, osteoclast surface area over bone surface area (Oc.S/BS) and the number of osteoclasts per bone perimeter (N.Oc/B.Pm) were measured by histomorphometric analysis. *, p < 0.05.

Fig. 5

AntagomiR-27a-treated mice have decreased bone mass and reduced osteoblast activity. Sham or OVX mice were injected i.v. with PBS, mut antagomiR-27a, or antagomiR-27a (three injections of 80 mg/kg/d in the first wk, and another injection on d 1-3 of the fourth wk) and bones were harvested at 6 wk after the first injection (n = 60 for each group). (A) Expression of Mef2c was measured by western blot analysis. (B) BMD of femurs was measured using a PIXImus densitometer. (C) Structure parameter bone volume/tissue volume ratio (BV/TV) was measured by micro-CT. (D) Bone formation parameters of mouse femur, bone formation rate over bone surface area (BFR/BS) and osteoblast surface area over bone surface area (Ob.S/BS) were measured by histomorphometric analysis. (E) Bone resorption parameters of mouse femur, osteoclast surface area over bone surface area (Oc.S/BS) and the number of osteoclasts per bone perimeter (N.Oc/B.Pm) were measured by histomorphometric analysis. *, p < 0.05.

Close modal

Reduced bone density is evident in the femurs of OVX mice compared with the healthy control mice. Next, bone formation parameters of mouse femurs were measured by histological analysis, these parameters included the bone formation rate per bone surface (BFR/BS) and the osteoblast surface per bone surface (Ob.S/BS). For the BFR/BS and the Ob.S/BS, a statistically significant decrease was detected between the PBS or mut antagomiR-27a controls and antagomiR-27a-treated OVX mice or control mice (p < 0.05) (Fig. 5D). Furthermore, a statistically significant increase in BFR/BS and Ob.S/BS was detected between the control mice and the OVX mice (p<0.05) (Fig. 5D). Bone resorption parameters of mouse femora were also measured by histological analysis, these parameters included the osteoclast surface per bone surface (Oc.S/BS) and the number of osteoclasts per bone perimeter (N.Oc/B.Pm). No statistically significant difference was detected between the PBS or mut antagomiR-27a controls and the antagomiR-27a-treated OVX mice or control mice, indicating that osteoclastic bone resorption was not affected (Fig. 5E). However, a statistically significant increase in BFR/BS and Ob.S/BS was detected for the OVX mice compared with the control mice for all constructs (p < 0.05).

The findings from this study demonstrate that miR-27a is essential for MSC differentiation along specific lineages, with expression levels being significantly increased during osteoblastogenesis and significantly decreased during adipogenesis. The balance between adipogenic and osteogenic differentiation is closely correlated with osteoporosis. Our data indicated that miR-27a is down-regulated in the development of osteoporosis.

We demonstrated that Mef2c is a direct target of miR-27a using a dual luciferase assay. A miR-27a binding site was also identified in the 3´UTR of the Mef2c gene. An inverse relationship between miR-27a expression and Mef2c mRNA expression in osteoporotic patients was shown. Mef2c is a transcription factor proposed to play a role in a variety of developmental processes including myogenesis, in particular, in maintaining the differentiated state of muscle cells [27], and cardiovascular development [28]. Much research has been carried out into the various regulatory processes involved in osteoblast differentiation. Transcription factors are central to this regulation, with RUNX2, Osterix and β-catenin known to be essential for osteoblast differentiation [29], and other transcription factors such as C/EBPβ, Smad1, Smad5 and Twist modulating the expression of RUNX2 [30]. This is the first report that Mef2c is also involved in osteoblast differentiation. Transcriptional control of gene regulation and regulation at the mRNA level by miRNAs are now known to both play a role in the control of skeletal development. The interplay between these two mechanisms, as demonstrated by the interaction between miR-27a and Mef2c expression reported in this study, remains to be fully elucidated and adds a further level of complexity to these control mechanisms. The cross-talk between these regulatory systems ultimately determines the differentiation status of the stem cells.

We also report that silencing of miR-27a decreased bone formation parameters, confirming the role of miR-27a in bone formation in vivo. Previous reports have suggested the role of miRNAs in osteoblast differentiation based on in vitro experiments in cell lines and increasing evidence demonstrates the regulatory role of miRNAs in osteoblast differentiation in vivo [19,31,32]. Inose et al. demonstrated that miR-206 regulated osteoblast differentiation in vivo in a similar manner to miR-27a [19]. Other miRNAs may yet to be reported that also play a role in this process.

In summary, miR-27a is essential for the shift from osteogenic differentiation to adipogenic differentiation in osteoporosis by targeting Mef2c. Overexpression of miR-27a in vivo leads to severe bone loss due to the impairment of osteoblast differentiation. It is therefore conceivable that the inhibition of specific miRNAs (e.g., using anti-miR-27a) may have therapeutic benefits for bone degenerative diseases such as osteoporosis.

LY participated in the design of the study; LY, LP, LC and WSG carried out the study; LY and JYC performed the statistical analysis; LY drafted the manuscript. All authors read and approved the final manuscript.

This work was sponsored by Natural Science Foundation of Shanghai (13ZR1433700).

The authors declare no conflict of interest.

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