Antibodies against PDCD4 (cat. no. ab45263), alkaline phosphatase (ALP; cat. no. ab83259), Bcl-2-associated X protein (Bax; cat. no. ab32503) and B-cell lymphoma 2 (Bcl-2; cat. no. ab32124) were purchased from Abcam (Cambridge, UK). The antibody against GAPDH (KC-5G5) was purchased from Zhejiang Kangchen Biotech Co., Ltd. (Wuhan, China). The secondary goat anti-rabbit (cat. no. BA1054) immunoglobulin (Ig)G-horseradish peroxidase-conjugated antibody was purchased from Boster Biological Technology (Pleasanton, CA, USA). A mimic (5′-UGGAAUGUAAGGAAGUGUGUGG-3′) and inhibitor (5′-CCACACACUUCCUUACAUUCCA-3′) for miR-206 was obtained from Chang Jing Bio-Tech, Ltd. (Changsha, China). A normal control (NC) mimic (5′-UUCUCCGAACGUGUCACGUT-3′) was purchased from Chang Jing Bio-Tech, Ltd. The Annexin V/propidium iodide (PI) apoptosis kit (cat. no. CCS012) was purchased from MultiSciences Biotech Co., Ltd. (Susteren, The Netherlands). Lipofectamine^™ 2000 (cat. no. 52887) reagent was obtained from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Cell-Light^™ 5-ethynyl-2′-deoxyuridine (EdU) Apollo^®488/567 in vitro Imaging kit was purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China; cat. no. C10327). RNAiso Plus (cat. no. 9109) was from Takara Bio, Inc. (Otsu, Japan). The Reverse Transcription (RT) kit and quantitative polymerase chain reaction (qPCR) agents were purchased from DBI^®Bioscience (www.xinghanbio.com/). A protein Concentration Determination kit (cat. no. 23227) was purchased from Thermo Fisher Scientific, Inc. A Dual Luciferase Assay kit (cat. no. E1980) was purchased from Promega Corporation (Madison, WI, USA).

Human osteoblast lineage hFOB1.19 and 293T cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in media consisting of 45% Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.), 45% F12 medium (Gibco; Thermo Fisher Scientific, Inc.), 10% fetal bovine serum and 1% mixed antibiotics (v/v) (penicillin/streptomycin) (R&D Systems, Inc., Minneapolis, MN, USA) at 37°C in an atmosphere of 5% CO₂ and 95% air. Cells from passage three to five were used for assays in the present study.

SANFH specimens were collected from 15 SANFH patients from February 2015 to March 2017 in the Department of Orthopedic Pathology in Baoan People's Hospital of the Southern Medical University (Shenzhen, China). A total of 15 bone specimens from patients undergoing total hip replacement for femoral neck fracture were collected as controls. All the cases involved in the present study had detailed information on the clinicopathological and prognostic characteristics. The present study was approved by Baoan People's Hospital of the Southern Medical University Ethics Committee. The Ethics Committee approved the associated screening, inspection and data collection of the patients, and all subjects signed a written informed consent form. All experiments were undertaken following the provisions of the Declaration of Helsinki.

Specific small interfering (si)RNA (50 nM) targeting PDCD4 (5′-UCAGUAUCUGCUCAUUUUCUATT-3′) and negative control (NC) siRNA (50 nM) (5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from Chang Jing Bio-Tech, Ltd. For the dual luciferase assay, wild type and mutant sequences of PDCD4 3′UTR were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and ligated into the psiCHECK-2 plasmid (Promega Corporation, Madison, WI, USA). Transfection was performed using Lipofectamine^™ 2000 according to the manufacturer's protocol. Twenty-four hours after transfection, cells were collected and subjected to subsequent experiments.

Total RNA from SANFH specimens or hFOB1.19 cells (normal, NC siRNA transfected, and PDCD4 siRNA transfected) was extracted using an RNA extraction kit according to the manufacturers' protocol. cDNA templates were obtained via RT using RNA according to the manufacturer's protocol. The final reaction mixture of volume 20 µl contained 10 µl Bestar^® SYBR-Green qPCR Master Mix and 0.5 µl each primer. PDCD4 forward, 5′-ACAGGTGTATGATGTGGAGGA-3′ and reverse, 5′-TTCTCAAATGCCCTTTCATCCAA-3′; miR-206 forward, 5′-ACACTCCAGCTGGGTGGAATGTAAGGAAGTGT-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; ALP forward, 5′-ACTGGGGCCTGAGATACCC-3′ and reverse, 5′-TCGTGTTGCACTGGTTAAAGC-3′. GAPDH was used as an internal reference, the primers were as follows: Forward, 5′-ATCGTGCGTGACATTAAGGAGAAG-3′ and reverse, 5′-AGGAAGGAAGGCTGGAAGAGTG-3′. U6 was used as an internal reference, the primers were as follows: Forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. A total of 1 µl cDNA template and 8 µl RNase free H₂O reaction volume was used. The thermocycling conditions for the amplification were as follows: A denaturation step at 94°C for 2 min, followed by 40 cycles at 94°C for 20 sec, 58°C for 20 sec and 72°C for 30 sec, and the reaction was stopped by a step at 25°C for 5 min. The melting curve was analyzed between 62°C and 95°C, and the relative expression levels of the target genes were calculated by a Real-Time PCR Detection system (Mx3000P; Agilent Technologies, Inc., Santa Clara, CA, USA) using the formula of 2^−ΔΔCq (20).

Total proteins were extracted using the Total Protein Extraction kit according to the manufacturer's protocol. The concentrations of the protein samples were determined according to the Bicinchoninic Acid assay. 30 µg protein from different samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis at 80 V for 2.5 h. The proteins were transferred onto polyvinylidene difluoride membranes and rinsed with Tris base buffer solution with Tween-20 (TBST). Following this, membranes were blocked with skimmed milk solution for 1 h at room temperature. Subsequently, membranes were incubated with one of the primary antibodies against PDCD4 (1:2,000) for 50 min, anti-Bax (1:1,000) for 25 min, anti-ALP (1:1,000) for 40 min, anti-Bcl-2 (1:1,000) for 25 min, and anti-GAPDH (internal reference; 1:1,000) for 30 min at 300 mA at room temperature and then incubated with peroxidase-conjugated secondary antibodies (1:20,000) for 40 min at room temperature. Blots were developed using the Beyo ECL Plus reagent (Beyotime Institute of Biotechnology, Haimen, China) and analyzed with the Gel Imaging system (WD-9413B; Beijing Liuyi Biotechnology Co., Ltd., Beijing, China) and the relative expression levels of different proteins were calculated with Bio-Rad Quantity One software version 4.6.3 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The modulation effect of miR-206 on PDCD4 was determined with dual luciferase assay in 293T cells (2×10⁵/ml): luciferase activity was detected by Dual Luciferase Assay kit according to the manufacturers' instruction 48 h following transfection of different combinations of plasmid and mimic. psi-CHECK2-PDCD4 and miR-206 mimic co-transfection was performed with the with Lipofectamine 2000. The fluorescence intensity was detected using a Microplate Reader (GloMax; Promega Corporation) and normalized in comparison with Renilla luciferase.

Exponentially growing hFOB1.19 cells (5×10⁵ cells/ml) were seeded into a 96-well plate. Then cells were incubated for 72 h (each treatment was administered nine times) and every 24 h, 10 µl/ml CCK-8 (Beyotime Insitute of Biotechnology) solution was added to selected wells and incubated at 37°C for 60 min. Optical density values at 450 nm were measured with a microplate reader (GloMax; Promega Corporation) and used to give a representative assessment of cell viability. hFOB1.19 cells were harvested following a 48-h culture and assessed using 5-ethynyl-2′-deoxyuridine (EdU) assay according to the manufacturer's protocol and the result was detected under a fluorescent microscope, magnification, 200x.

Immunofluorescence detection was conducted as described in a previous study (21). Briefly, paraformaldehyde-fixed cells were permeabilized with 0.1% Triton X-100 for 30 min. Following blocking in 10% goat serum (Thermo Fisher Scientific, Inc.) for 15 min, primary rabbit polyclonal antibodies against different proteins of interest (PDCD4) (1:500) were then added, and the cells were incubated overnight at 4°C. Subsequently, cells were incubated with Cy3-labeled secondary antibody (1:1,000) (cat. no. ab6939; Abcam) for 1 h in the dark. Thereafter, the cells were stained with 4,6-diamino-2-phenyl indole for 5 min at room temperature. The results were imaged with a fluorescent microscope (BX53; Olympus Corporation, Tokyo, Japan) at magnification, ×40.

The morphological alterations of the cellular nuclei due to apoptosis were detected using a Hoechst staining kit (20 µM) according to the manufacturer's protocol. The results were observed under a fluorescence microscope under 460 nm; magnification, ×120.

Apoptotic process was detected using an Annexin V/PI apoptosis kit according to the manufacturer's protocol. The apoptotic rates were analyzed using a FACScan flow cytometer (Accuri C6; BD Biosciences). The total apoptotic rate was equal to the sum of the late apoptotic rate (UR, upper right quadrant-advanced stage apoptosis) and the early apoptotic rate (LR, lower right quadrant-prophase apoptosis).

All the data were expressed as the mean ± the standard deviation (n=3). The association between expression of miR-206 and PDCD4 at mRNA level was analyzed with Pearson correlation analysis. The difference between groups was analyzed using Student's t-test or one-way analysis of variance followed by the Least Significant Difference. All the statistical analyses were conducted using SPSS version 19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

A total of 15 SANFH samples were collected to investigate the association between the expression of miR-206 and PDCD4. As presented in Fig. 1A, the expression of miR-206 was significantly increased in SANFH samples compared with the control samples (P<0.001). However, the expression of PDCD4 was significantly inhibited at the mRNA and protein level (P<0.01; Fig. 1B and C, respectively). In addition, it was demonstrated that miR-206 was negatively correlated with PDCD4 at the mRNA level (R=0.633, P<0.01; Fig. 1D), indicating a potential interaction between the two factors.

The interaction between miR-206 and PDCD4 was analyzed with a dual luciferase assay. As presented in Fig. 2, the co-transfection of miR-206 and the wild type promoter sequence of PDCD4 significantly decreased the relative luciferase activity in 293T cells (P<0.05). Given that miR-206 and the mutant promoter sequence of PDCD4 had no effect on relative luciferase activity, the results of the dual luciferase assay indicated the that miR-206 bound directly and specifically to the promoter sequence of the PDCD4 gene, which exerted a negative effect on the expression of PDCD4 at the transcriptional level.

The function of miR-206 in osteoblastogenesis was assessed by altering the expression of miR-206 in a bilateral pattern. Both a specific mimic and inhibitor of miR-206 were stably introduced into the human osteoblast lineage hFOB1.19. Based on the results of the CCK-8 assay, the cell viability of miR-206 overexpressing cells was significantly decreased, while the viability of the cells treated with an miR-206 inhibitor significantly increased compared with the control (P<0.05; Fig. 3A). Consistent with the results of the CCK-8 assay, a greater number of EdU positive cells (stained red) were detected in the cells treated with the miR-206 inhibitor while fewer was detected with miR-206 overexpressing cells (Fig. 3B). The cells overexpressing-miR-206 demonstrated a significantly increased level of apoptosis as illustrated by Hoechst33258 staining and flow cytometry: A larger proportion of Hoechst33258 positive cells (bright blue), and significantly increased apoptotic rate were detected (P<0.01; Fig. 3C and D). However, inhibition of miR-206 in hFOB1.19 cells had no influence on the cell apoptotic process compared with the NC group (Fig. 3C and D).

At the molecular level, transfection of an miR-206 mimic downregulated the expression of PCDCD4 (Fig. 4A-C). Additionally, ALP was also suppressed by transfection with an miR-206 mimic (Fig. 4A and B), indicating the impaired proliferation and differentiation potential of hFOB1.19 cells as a result of miR-206 overexpression. The level of the pro-apoptotic factor Bax was increased while the level of anti-apoptosis factor Bcl-2 was suppressed in miR-206 overexpressing cells (Fig. 4B). For cells transfected with the miR-206 inhibitor, the expression of Bax was decreased and the expression of PCDCD4 and Bcl-2 was increased (Fig. 4B and C), representing an inhibited apoptotic process in hFOB1.19 cells, which explained the augmented cell viability by miR-206 knockdown.

The involvement of PDCD4 in osteoblastogenesis had been previously reported (22), but the exact function of the factor on bone metabolism was neglected. The effect of PDCD4 and miR-206 on the differentiation potential, and apoptosis was investigated (Fig. 5). In the present study, the expression of PDCD4 was inhibited in miR-206 knockdown hFOB1.19 cells, which resulted in significantly decreased expression of ALP (P<0.001; Fig. 5A). It was demonstrated that the knockdown of PDCD4 significantly induced cellular apoptosis in a similar pattern to that of miR-206 overexpression (P<0.01; Fig. 5B and D). In addition, knockdown of PDCD4 induced the expression of Bax while the expression of ALP and Bcl-2 was suppressed (Fig. 5C), which relieved the hFOB1.19 cells of the effect of miR-206 inhibition. The above results indicated that the pro-SANFH function of miR-206 depended on the suppression of PDCD4 in osteoblasts.