The experiments were performed using 200 8-week-old female Wistar rats (BetterBiotechnology Co., Ltd., Nanjing, China) weighing between 180 and 200 g. Throughout the experiment, the animals were housed in groups in a room with controlled temperature (22°C) and humidity (50%) with a 12 h light-12 h dark cycle. Standard rat chow (BetterBiotechnology Co., Ltd.) and tap water were available ad libitum. All procedures involving the animals were approved by the ethics committee of China Medical University (Shenyang, China).

The OVX was performed under isoflurane (10%, 0.35 ml/100 g) anesthesia. A single dorsal incision was made in the skin and, through this, two lateral incisions were made in the muscle layer ~0.5 cm below each kidney. The ovaries were extruded through the incision, ligated off and removed. At 20 days post-surgery, a 500 mg/kg dose of RESV was prepared by mixing RESV (Sigma-Aldrich, St. Louis, MO, USA) with deionized water at a ratio of 3:7, and was administered to the OVX female rats through tail intravenous injection. The OVX rats were divided into four groups (n=10 per group) as follows: Untreated rats; OVX rats OVX rats with phosphate-buffered saline (PBS; Sigma-Aldrich); and OVX rats with RESV. The subcutaneous fat and the uterus were also harvested, rinsed with physiological saline, and weighed.

The BMD was assessed using dual-energy X-ray absorptiometry with a Hologic QDR-4500A X-ray bone densitometer (Hologic, Inc., Danbury, CT, USA). The total body BMD was assessed on the 30th and 60th day following the administration of RESV.

The concentrations of calcium, phosphorus and creatinine in the serum and urine were measured using a commercial kit (Quanti Chrom™Bioassay Systems, Hayward, CA, USA). The levels of serum alkaline phosphatase (ALP) and osteocalcin were measured using a commercial ALP kit (Asan Pharm, Seoul, Korea) and Rat-MID™ Osteocalcin EIA kit (Immunodiagnostic systems Inc., Stoughton, MA, USA), respectively. The level of urinary deoxypyridinoline (DPD) was measured using a METRA™ DPD EIA kit (Quidel Corporation, San Diego, CA, USA). All experiments were performed according to the manufacturer’s instructions and the results were analyzed using a microplate reader (Bio-Rad Laboratories, Inc., Richmond, CA, USA). The values for DPD were adjusted for volume by determining the level of urinary creatinine (Quanti Chrom™ Bioassay Systems), with concentrations presented as nmol DPD/mmol creatinine.

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and further purified using an mirVana miRNA isolation kit (Life Technologies, Grand Island, NY, USA). Microarray analysis was performed using an Agilent Whole Rat Genome Array (Agilent, Santa Clara, CA, USA), according to the manufacturer’s instructions. The integrity of the total RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent). cDNA was synthesized from RNA by reverse transcription using the PrimeScript™ II 1st strand cDNA synthesis kit (cat. no. 6210B; Takara Bio Inc., Dalian, China). The cDNA and biotinylated cRNA were synthesized and hybridized to the array. The slides were scanned using an Agilent Microarray Scanner (cat no. G2565BA; Agilent) and Feature Extraction software 10.7 (Agilent) with default settings. The raw data were normalized by Quantile algorithm, Gene Spring software 11.0 (Agilent).

The rats were sacrificed using ketamine (70 mg/kg) and xylazine (6 mg/kg) purchased from Sigma-Aldrich. The femur tissues were obtained from the rat models and lysed in 20 mM Tris-HCl buffer (pH 7.4), containing 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 50 mM NaF, 1 mM Na₃VO₄, 1 mM Na₂MoO₄, 10 μm leupeptin (Roche, Basel, Switzerland). The protein concentration was quantified using a bicinchoninic protein assay kit (CW Biotech, Beijing, China). The crude lysates were then centrifuged at 14,000 × g for 10 min, following which the cleared lysates were collected and were separated by 8% SDS-polyacrylamide gel electrophoresis (Sigma-Aldrich) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% milk-Tris-buffered saline with 5% Tween-20 (Sigma-Aldrich) and incubated with primary antibodies at room temperature overnight. Rabbit polyclonal anti-runt-related transcription factor (RUNX)2 (1:200; cat. no. sc-10758) and mouse monoclonal anti-β-actin (1:200; cat. no. sc-47778) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The secondary antibodies were purchased from Beyotime Biotechnology (Beijing, China). The secondary antibodies included anti mouse IgG (cat. no. A0216), anti rabbit IgG (cat. no. A0239) or anti goat IgG (cat. no. A0181). The membranes were incubated with the secondary antibodies for 2 h at room temperature. The membranes were then developed and visualized using a Pierce ECL kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Densitometric quantification for western blotting was performed using Scion 4.03 image software (Scion Corporation, Bethesda, MD, USA).

The target gene information of mmu-miR-338-3p was analyzed using microRNA TargetScan software (http://www.microrna.org/mammalian/index.html). The minimum free energy predicted for hybridization was determined by BibiServ analysis (http://bibiserv.techfak.uni-bielefeld.de/genefisher2).

Human osteoblast (HOB) cells were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and maintained in osteoblast basal media (Lonza, Walkersville, MD, USA) supplemented with fetal bovine serum (Lonza).

The anti-miRNA inhibitor (anti-miR-338-3p) and negative control miRNA (anti-control) were purchased from Ambion Life Technologies, Carlsbad, CA, USA). For transfection, the cells were plated at 70–80% confluency in a 6-well dish. After 24 h at room temperature, the cells were transfected with 1 μg chemically-synthesized RNA (GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer’s instructions.

The cells were washed with PBS, fixed with 4% formaldehyde in PBS for 20 min, and finally permeabilized with 0.2% Triton X-100 solution (Beyotime Biotechnology) for 5 min at room temperature. The cells were incubated with anti-RUNX2 antibody for 1 h, washed three times with PBS and then incubated with the appropriate Alexa Fluor 594-conjugated secondary antibody The cells were then washed three times in PBS. The slides were mounted with VECTASHIELD mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). For every cover-slip, the cells were visualized and images were captured in five random fields using an Olympus CX71 fluorescence microscope (Olympus Corporation, Tokyo, Japan).

The HOBs were plated at a density of 1×10³ cells/well in 96-well plates. The mitogenic activity following treatment with RESV was assayed using a colorimetric 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymetho xyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, WI, USA). Following treatment of the different groups, 20 μl MTS was added to each well for 4 h at room temperature, and the light absorbance at 490 nm was measured using a spectrophotometric microplate reader (Model 680; Bio-Rad Laboratories, Inc.).

The cells were seeded in 6-well plates, in triplicate, at a density of 3×10³/cm². Alizarin Red S staining, which detects calcium deposition, was used as an indicator of mineralization. The cells were rinsed in PBS and fixed in 70% ice-cold ethanol prior to staining with 40 mM Alizarin Red S (pH 4.2; Sigma-Aldrich) for 10 min at room temperature. The calcium content was then quantified by measuring the level of Alizarin Red S staining, which was bound to the mineralizing nodules.

Dual-luciferase reporter assays were performed in the HOB cells to determine the binding site of miR-338-3p within the 3′UTR of RUNX2. Briefly, a pMIR-reporter (Signosis, Inc., Santa Clara, CA, USA), containing either the wild-type or mutant 3′-UTR was cotransfected with phRL-TK (Signosis, Inc.) into the HOB cells. At 48 h post-transfection, the cells were harvested and analyzed using a dual-luciferase reporter assay (Promega), according to the manufacturer’s instructions. Briefly, the cell extract was subjected to dual-luciferase assay as measured by the Xenogen IVIS 200 imaging system (Caliper Life Sciences, Hopkinton, MA, USA). The data were presented as luciferase activity/RL-TK activity.

qPCR was performed using specific primers. qPCR analysis was performed on an ABI prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA), using SYBR Green PCR master mixture (Takara Bio Inc.). The PCR conditions were as follows: One cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. The following primer sets were used: RUNX2, sense 5′-ATGGCCTGGTCCATCTCCAC-3′ and antisense 5′-GGCAGAGGTGAAAAAGTTGC-3′ and GAPDH, sense 5′-CACTGGCGGTGCAACAAGA-3′ and antisense 5′-CATGGGTGGAATCATATTGGAA-3′. The miRNA-338-3p and U6 primers were purchased from Qiagen (Hilden, Germany). Relative quantification was calculated using the 2^−∆∆Ct method, where ∆∆Ct = Ct(RUNX2) - Ct(GAPDH) (10). The expression level of the control group was considered “1”.

A total of 19 nucleotide sequences of miR-338-3p from a wide range of organisms were obtained from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/). Multiple sequence alignments were performed using the ClustalW programme (11). To determine the phylogenetic associations between these sequences, maximum likelihood, neighbor-joining and Bayesian Markov chain Monte Carlo approaches were used to infer three individual trees.

The data are presented as the mean ± standard deviation Statistical analysis was performed using a two-tailed t-test using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated at least three times.

The baseline body weights of the animal groups were similar (Table I), however, the final body weights and subcutaneous fat weights were significantly higher in the OVX group and the OVX + PBS groups, compared with the untreated group and the OVX + RESV groups (Tables I and II), while the uterus weight was significantly lower in the OVX group compared with the OVX + RESV group (Table III). The body weights and levels of subcutaneous fat decreased significantly in the ratstreated with RESV compared with those in the OVX-only group (Tables I and II).

The BMD was significantly lower in the OVX group compared with the untreated group, and OVX + RESV increased the BMD (Table IV). When comparing the OVX group to the untreated group, the calcium/phosphate ratio and the levels of serum calcium were significantly lower, however, the levels of serum ALP, urinary calcium excretion and urinary DPD/creatinine were significantly higher (Tables V–X). The rats treated with RESV had significantly increased levels of serum osteocalcin and calcium/phosphate ratios, however, they exhibited decreased levels of urinary calcium and DPD excretion compared with the rats in the OVX group (Tables V–X). The serum calcium level to increase and the levels of serum ALP and serum phosphorus tended to decrease in the rats that treated with RESV (Tables V–X).

To identfiy miRNAs, which may have been involved in RESV-induced differentiation in the OVX group, array analysis was used to screen for miRNAs, which were differentially expressed between the OVX group and the OVX + RESV group (Fig. 1). Changes of several miRNAs were identified, of which miR-338-3p was examined in the present study as it exhibited the most marked change in expression. qPCR analysis confirmed that miR-338-3p was significantly decreased in the OVX + RESV treatment compared with the OVX group (Fig. 2A). Furthermore, qPCR and western blot analysis were performed in order to determine the mRNA and protein levels of RUNX2 in each group, respectively (Fig. 2B and C). The mRNA expression of RUNX2 was negatively correlated with that of miR-338-3p (rs=−0.7518; P<0.05; Fig. 2D).

TargetScan was used to identify the potential targets of miR-338-3p. Runx2 was identified as a target of miR-338-3p (Fig. 3A). BibiServ analysis determined the free energy for the RUNX2 3′UTR region and miR-338-3p hybrid was ~−11.2 kcal/mol (Fig. 3B). The relative luciferase activity results revealed that the RUNX2 reporter was suppressed by miR-338-3p (Fig. 3C). These results demonstrated that miR-338-3p specifically targeted the 3′UTR region of RUNX2.

The present study also investigated the effects of miR-338-3p knockdown in HOB cells, by transfection of the HOB cells with miR-338-3p inhibitor. As shown in Fig. 4A, the results of qPCR confirmed the downregulation of endogenous miR-338-3p in the HOB cells following transfection (P<0.05). Immunofluorescence analysis revealed an increase in the expression of RUNX2 in the HOB cells following transfection (Fig. 4B). Compared with the untransfected cells, a significant increase was observed in the proliferation rate of the HOB cells (P<0.05; Fig. 4C). miR-338-3p knockdown also promoted in vitro calcium deposition in the HOB cells (Fig. 4D). Finally, multiple sequence alignment and phylogenetic analysis were performed, the results of which confirmed miR-338-3p as is a highly conserved microRNA (Fig. 5A and B).