The human osteoblastic cell line hFOB, OS cell lines (U2OS, MG-63, and SAOS-2) and 293T cells were purchased from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified 5% CO₂ atmosphere.

A total of 48 OS tissues and self-matched non-tumor tissues were collected from 48 patients with OS between February 2014 and September 2015. In total, 40 cases were <18 years old and 8 cases were >18 years old, and 22 cases were women and 26 cases were men. All tissue samples were obtained via surgical resection and stored in liquid nitrogen until use in further analyses. All OS tissues were pathologically confirmed, and OS specimens that received chemotherapy, radiotherapy, or other types of therapy were excluded from the present study. The collection and the use of all tissue samples in the present study were approved by the Research Ethics Committee of Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine (Wenzhou, China). Written informed consent was obtained from all participating patients.

Total RNA was isolated from OS cell lines, OS tissues and self-matched adjacent normal tissues using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Purified mRNA and miRNAs were quantified by a RT-qPCR assay using an All-in-One^™ miRNA RT-qPCR Detection kit (GeneCopoeia, Inc., Rockville, MD, USA) according to the manufacturer's protocol. All primers are listed in Table I. The reverse primer sequence of miR-542-3p was provided by All-in-one miRNA RT-qPCR Detection kit (GeneCopoeia, Inc.). U6 small RNA was used as an internal control for normalization, miR-542-3p expression were calculated as 2^{−[(Ct miR-542-3p)-(Ct U6)]}, where Ct represents the threshold cycle for each transcript.

RNA oligos were chemically synthesized and purified by Shanghai Genepharma Co., Ltd., (Shanghai, China). The sense sequence of the human miR-542-3p mimics was 5′-UGUGACAGAUUGAUAACUGAAA-3′, and the antisense sequence was 5′-UUUCAGUUAUCAAUCUGUCACA-3′. The sequences of the negative control oligonucleotides were 5′-AAUUCUCCGAACGUGUCACTT-3′ and 5′-GUGACACGUUCGGAGAAUUTT-3′. In total, 1×10⁵ cells were seeded in a 6-well plate, and the transfections were performed with INTERFER in reagent (Polyplus-transfection SA, Illkirch, France) according to the manufacturer's protocol. The final concentration of miRNA was 50 nM.

To construct a pGL3-Smad2 vector, the sequence of Smad2 mRNA was amplified (primers are listed in Table I). The Smad2 fragments were inserted into pGL3 by the designed cutting sites: KpnI and XhoI. In total, 1×10⁵ cells were seeded in a 6-well plate, the transfections were performed with Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The final concentration of plasmids was 100 ng.

The pMIR-Smad2 luciferase reporter vector was constructed by cloning a human Smad2 mRNA sequence into a pMIR-Report construct (Ambion; Thermo Fisher Scientific, Inc.). A 60-bp Smad2 mRNA fragment (5,084–5,143 bp of Smad2 mRNA), which is the predicted target of miR-542-3p, was amplified and cloned into the luciferase reporter via SpeI and HindIII sites. All sequences are listed in Table I. Luciferase reporter assays were performed as follows: 5×10³ 293T cells were seeded in a 96-well plate, then co-transfected with 50 nM single-stranded miR-542-3p mimics or negative control oligonucleotides, 10 ng of pMIR-Smad2 luciferase reporter, pMIR luciferase reporter and 3 ng of pRL-TK (Promega Corporation, Madison, WI, USA) using the JetPRIME^® reagent (Polyplus-transfection SA). Cells were collected 36 h after transfection and analyzed using a Dual-Luciferase Reporter assay system (Promega Corporation). pRL-TK was cotransfected as an internal control to correct the differences in both transfection and harvest efficiencies. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity.

The proliferation of OS cells was measured by MTT assay. A total of 5,000 cells were seeded into each well of 96-well plates and transfected with miR-542-3p mimics or negative control oligonucleotides at a final concentration of 50 nM. At 24, 48 and 72 h after transfection, the medium was replaced with 100 µl fresh medium containing MTT (0.5 mg/ml), and the plates were incubated for 4 h at 37°C. The medium was replaced by 100 µl DMSO (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and plates were agitated at room temperature for 10 min. The absorbance was measured at 570 nm.

Cell apoptosis analyses were performed using a Phycoerythrin-Annexin V Apoptosis Detection kit I (BD Pharmingen; BD Biosciences, San Jose, CA, USA). For cell apoptosis analysis, 8×10⁵ cells were seeded in each well of 6-well plates. At 78 h after transfection, cells were harvested and labeled with Annexin V for 15 min. Subsequently, 50 µg/ml propidium iodide was added for 1 h at 37°C to each sample prior to flow cytometry using the BD LSR II (BD Biosciences).

The possible targets of miR-542-3p were screened by the DIANA-TarBase web platform (version 7; http://diana.imis.athena-innovation.gr/), which included >500,000 experimentally confirmed miRNA-mRNA interactions utilizing cell types from 24 species (18).

MG-63 and U2OS cells were washed with pre-chilled PBS three times. The cells were then solubilized in 1% Nonidet P-40 lysis buffer [20 mM Tris, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM CaCl2, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and phosphatase inhibitor cocktail II (5 mg/ml, Sigma-Aldrich; Merck KGaA)] for 0.5 h at 4°C, and protein concentration was determined using a bicinchoninic acid assay (BCA Protein Assay Kit, Pierce; Thermo Fisher Scientific, Inc.). Proteins (40 µg) were separated on a 12% SDS-PAGE gel and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was blocked with 5% non-fat milk in PBS with 0.1% Tween-20 for 2 h and washed three times with PBS with 0.1% Tween-20 at 4°C, then incubated with anti-Smad2 antibody (1:1,000 dilution) or anti-β-actin antibody (1:5,000 dilution) (Sigma-Aldrich; Merck KGaA). Following extensive washing, a goat anti-mouse secondary antibody (1:1,000 dilution) (Pierce; Thermo Fisher Scientific, Inc.) was added to the system. The proteins were detected using enhanced chemiluminescence reagents (Pierce; Thermo Fisher Scientific, Inc.). Protein bands were quantified using Pearson's correlation coefficient analysis (LabWorks software version 4.0; Image Acquisition; UVP, Inc., Upland, CA, USA).

All statistical analyses were performed using the SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Data of miR-542-3p expression levels in OS specimens compared to matched adjacent normal tissues were subjected to analysis by paired Student's t-test. Data of miR-542-3p expression levels in several cell lines and MTT analysis were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

In order to identify the role of miR-542-3p in OS carcinogenesis, the expression levels of miR-542-3p in 48 OS samples and three OS cell lines were analyzed. Total RNAs were extracted from OS tissues and adjacent normal tissues. The expression levels of miR-542-3p were analyzed using RT-qPCR and normalized to an endogenous control (U6 RNA). As shown in Fig. 1A, the expression of miR-542-3p was significantly decreased in OS tissues vs. adjacent normal tissues (0.0066±0.0033 vs. 0.0141±0.0040). The results also demonstrated that the expression of miR-542-3p was significantly downregulated in OS cell lines, U2OS, MG-6 and SAOS-2, compared with which in human osteoblastic cell line hFOB (Fig. 1B). This indicates that miR-542-3p may function as an oncosuppressor gene in OS carcinogenesis.

To further investigate the effect of miR-542-3p on the proliferation ability of OS cells, gain of function studies were performed in two human OS cell lines, MG-63 and U2OS. miR-542-3p mimics or negative control oligonucleotides were transiently transfected into MG-63 and U2OS cells. Expression of miR-542-3p was detected by RT-qPCR. The results confirmed that expression levels of miR-542-3p in MG-63 and U2OS cells were significantly increased following transfection of miR-542-3p mimics (Fig. 2A). Next, the functional role of miR-542-3p in cell proliferation in OS cells was investigated by performing MTT assays. Restoration of miR-542-3p expression in MG-63 and U2OS cells resulted in significant suppression of cell proliferation (Fig. 2B). Using flow cytometry, the influence of overexpression of miR-542-3p on apoptosis in U2OS and MG63 cells was evaluated following transfection of miR-542-3p mimics. Overexpression of miR-542-3p significantly increased the rate of apoptosis in MG-63 (63.77 vs. 32.07% in the control group; P<0.01) and U2OS cells (54.03 vs. 27.9% in the control group; P<0.01; Fig. 2C).

After confirmation of function action of miR-542-3p in OS cells, the underlying mechanism of the effects of miR-542-3p in OS was investigated. Recently published DIANA-TarBase v7.0 data included over half a million experimentally confirmed miRNA-mRNA interactions utilizing cell types from 24 species (18). Following this analysis, many potential miR-542-3p targeting genes were identified (data not shown). Among these targeting genes, the present study focused on Smad2, which is a member of the Smad family. Smad2 has been previously identified in the regulation of cell proliferation and in apoptosis as a key element in TGF-β signaling (19,20). In addition, mirPath (http://www.microrna.gr/miRPathv2) was used to perform the pathway analysis of the target genes of miR-542-3p. Enrichment analysis identified the 12 most significant pathways, which included the ‘TGF-β signaling pathway’ (P=0.003; Table II).

Consequently, the present study established whether Smad2 was a genuine target of miR-542-3p by performing a set of experiments. To confirm whether miR-542-3p regulated the expression of Smad2 gene, a luciferase reporter assay was performed in 293T cells. As indicated in Fig. 3A, a targeting sequence of Smad2 mRNA was cloned into a luciferase reporter plasmid. These luciferase reporter plasmids were co-transfected into 293T cells with miR-542-3p mimics or negative control oligonucleotides. The luciferase activities in transfected cells were measured after 36 h. As shown in Fig. 3B, overexpression of miR-542-3p caused a significant decrease in luciferase activity in cells transfected with the reporter plasmid containing the miR-542-3p-targeted sequence of Smad2 mRNA, whereas overexpression of miR-542-3p produced no significant change in luciferase activity in cells transfected with the reporter plasmid without the targeted sequence of Smad2. Subsequently, the present study investigated whether endogenous Smad2 in OS cells was similarly regulated. MG-63 and U2OS cells were transfected with miR-542-3p mimics or negative control oligonucleotides. The mRNA and protein levels of Smad2 were examined by RT-qPCR and western blotting, respectively. The level of Smad2 protein was consistently and substantially downregulated by miR-542-3p; however, the level of Smad2 mRNA was not affected by miR-542-3p (Fig. 3C and D). Finally, the expression levels of miR-542-3p and Smad2 were analyzed in four representative OS tumor tissues and adjacent non-tumor tissues by RT-qPCR and western blotting. The results confirmed that OS tumor tissues with low expression of miR-542-3p exhibited markedly higher Smad2 expression compared with adjacent non-tumor tissues (Fig. 3E and F).

Taken together, these results demonstrated that Smad2 is a direct target of miR-542-3p in OS, and that miR-542-3p directly regulates Smad2 expression at the protein level.

In order to further confirm that miR-542-3p exerts tumor-suppressor activity in OS pathogenesis through targeting Smad2, gain of function and rescue experiments were performed in OS cells. It was investigated whether the proliferation of OS cells was further promoted following transfection with Smad2 cDNA, and whether this effect could be attenuated by miR-542-3p mimics. Smad2 cDNA was transiently transfected into MG-63or U2OS cells with and without miR-542-3p mimics. Subsequently cell proliferation assays were performed.

As hypothesized, ectopic expression of Smad2 in MG-63 cells enhanced the accumulation of Smad2, while restoration of the miR-542-3p expression in MG-63 cells markedly inhibited the expression of Smad2 (Fig. 4A). Furthermore, restoration of miR-542-3p inhibited the Smad2 expression in U2OS cells (Fig. 4B). In conjunction with these results, the overexpression of miR-542-3p significantly attenuated the Smad2-induced increase in cell proliferation in MG-63 and U2OS cells (Fig. 4C and D). Taken together, these results demonstrated that restoration of miR-542-3p was able suppress the growth and proliferation of OS cells through directly targeting Smad2.