The OS cell lines used in the present study, including 143B, U2OS, KHOS-240S, Saos-2 and MG-63, and the normal cell line (hFOB1.19) were purchased from The Shanghai Institute of Cell Biology (Shanghai, China). The cells were maintained in RPMI-1640 medium supplemented by 10% fetal bovine serum (FBS), streptomycin (100 µg/ml) and penicillin (200 U/ml; all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in 5% CO₂ at 37°C. A total of 87 surgically resected OS specimens and adjacent normal tissues were acquired from patients at Beijing Rehabilitation Hospital of Capital Medical University between June 2013 and August 2016. All patients signed formal consent forms. The experiments involving human specimens were reviewed and approved by the Ethics Committee of Beijing Rehabilitation Hospital of Capital Medical University.

For CCND1 overexpression, the coding sequence of CCND1 was constructed into pCDNA3-myc vector (pCDNA3-myc-CCND1). The miR-466 mimics, miR-466 inhibitor and negative controls were synthesized and purchased from Sigma-Aldrich; Merck KGaA. Corresponding plasmids were transfected into 143B and U2OS cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. After incubation for 48 h, cells were used for further analysis.

For cell proliferation assays, the viable cells were tested by Cell Counting Kit-8 (CCK-8) assay kit according to the manufacturer's instructions. In brief, cells were grown in 96-well plate with 1×10⁴ per well and incubated in 37°C with 5% CO₂ until cell confluent rate reached 70%. After transfected with plasmid for 48 h, cells were still incubated for 24, 48, 72 and 96 h. 10 µl CCK8 solution was seed into each well. The absorbance at 490 nm was measured with SUNRISE Microplate Reader (Tecan Group, Ltd., Mannedorf, Switzerland).

Total RNA was extracted from tissues and cells using the RNeasy Plus mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. RNA was then reversely transcribed into complementary DNA using the PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions. For quantification, PCR was performed using a miRNA Q-PCR Detection kit (GeneCopoeia, Inc., Rockville, MD, USA) on an ABI 7500 thermocycler (Thermo Fisher Scientific, Inc.). U6 or GAPDH was used as an internal reference. The relative expression of mRNA was determined using the 2^−∆∆Cq method (16). The primer sequences were as follows: miR-466 (5′-AACGAGACGACGACAGAC-3′ and 5′-ATACACATACACGCAACACACAT-3′), U6 (5′-AACGAGACGACGACAGAC-3′ and 5′-GCAAATTCGTGAAGCGTTCCATA-3′), CCND1 (5′-TGAGGGACGCTTTGTCTGTC-3′ and 5′-GCCTTTGGCCTCTCGATACA-3′) and GAPDH (5′-ATGTTGCAACCGGGAAGGAA-3′ and 5′-AGGAAAAGCATCACCCGGAG-3′).

Total proteins were isolated by radioimmunoprecipitation (RIPA) assay buffer (Wlaterson, Barcelona, Spain). Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Wlaterson). A total of 50 µg protein was separated by 12% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 10% skim milk (w/v) at room temperature for 2 h. Target proteins were probed with specific antibodies against Cyclin D1 (1:2,000; cat. no. 2978; Cell Signaling Technology, Inc., Danvers, MA, USA) and GAPDH (1:5,000; cat. no. 5174; Cell Signaling Technology, Inc.), followed by incubation with horseradish peroxidase conjugated goat-anti-rabbit second antibody (1:5,000; cat. no. 7074; Cell Signaling Technology Inc.). The blots were detected with the Enhanced Chemiluminescence western blot detection kit (Pierce; Thermo Fisher Scientific, Inc.).

Each experiment was repeated at least three times. All data are expressed in terms of means ± SD. The Kaplan-Meier method was used to calculate the survival curve, and log-rank test to determine statistical significance. The differences between groups were analyzed using Two-tail Student's t-test and ANOVA followed by Tukey's post hoc test. Pearson correlation coefficient analysis was used to determine the expression correlation between CCND1 and miR-466. Chi-square test was used for analysis of correlation between miR-466 expression and clinicopathological characteristics. P<0.05 was considered to indicate a statistically significant difference.

To explore the function of miR-466, we determined its expression in OS tissues and adjacent normal tissues by RT-qPCR. The results indicated that miR-466 was significantly downregulated in OS tissues (n=87) compared with adjacent normal tissues (n=87; Fig. 1A). Consistently, miR-466 expression was also downregulated in OS cell lines, including 143B, U2OS, KHOS-240S, UMR-106, Saos-2, and MG-63 cells compared with hFOB1.19 cells (Fig. 1B). Then, we divided these OS samples into two groups based on miR-466 expression level and analyzed the correlation between miR-466 expression and clinicopathological characteristics. We found that miR-466 expression in OS tissues was negatively correlated with differentiation, tumor metastasis, and tumor, node, and metastasis (TNM) stage, whereas no significant correlation was observed with age and gender (Table I). Furthermore, we performed Kaplan-Meier curve to determine the relationship between miR-466 expression and prognosis of patients with OS. The results showed that high expression of miR-466 in patients with OS was linked to high survival rate (Fig. 1C). Overall, our data indicated that miR-466 was downregulated in OS tissues and correlated with patients' severity and prognosis, implying that miR-466 may exert an important role in OS progression.

To investigate the physiological functions of miR-466, we selected 143B and U2OS cells for the following experiments. We effectively knocked down or overexpressed miR-466 in 143B and U2OS cells by transfection with miR-466 mimic or inhibitor (Fig. 2A). Then, we performed CCK8 assays to measure cell proliferation. We found that miR-466 overexpression significantly inhibited the proliferation of 143B and U2OS cells, and vice versa (Fig. 2B and C). To explore whether the reduced cell proliferation is induced by aberrant cell cycle, we stained 143B and U2OS cells with PI and performed FACS analysis. The results indicated that miR-466 overexpression significantly increased the percent of cells in G0/G1 phase and decreased cells in S phase, and vice versa (Fig. 2D and E). Furthermore, we found that miR-466 overexpression remarkably promoted the percentages of apoptotic 143B and U2OS cells (Fig. 2F). Collectively, these results indicated that miR-466 suppressed OS cell proliferation and induced apoptosis.

Then, we investigated the molecular mechanism of miR-466. Through bioinformatics analysis, we found that CCND1 encoding Cyclin D1 was a potential target of miR-466. A potential binding site of miR-466 in the 3′-UTR region of CCND1 mRNA was observed (Fig. 3A). Then, we constructed CCND1-3′-UTR-WT or mutant (MUT) luciferase reporter plasmid for luciferase reporter assays. The results demonstrated that miR-466 overexpression significantly inhibited the luciferase activity in 143B and U2OS cells transfected with CCND1-3′-UTR-WT (Fig. 3B and C). However, mutation of the potential binding site in the 3′-UTR region of CCND1 mRNA abrogated the effect of miR-466 transfection (Fig. 3B and C). Moreover, RT-qPCR and Western blot results showed that miR-466 overexpression inhibited the mRNA and protein levels of CCND1 in 143B and U2OS cells (Fig. 3D and E). We also found that the miR-466 expression was reversely correlated with that of CCND1 in OS tissues (Fig. 3F). Moreover, we found that CCND1 was also highly expressed in OS tissues compared with adjacent normal tissues (Fig. 3G). Overall, above results demonstrated that CCND1 is a direct target of miR-466 in OS cells.

To further validate that CCND1 is important for the function of miR-466 in OS cells, we performed rescue experiments. We restored the protein levels of CCND1 in 143B and U2OS cells transfected with miR-466 mimics. We successfully overexpressed CCND1 in 143B and U2OS cells (Fig. 4A). And western blot results also indicated that CCND1 was significantly upregulated in 143B and U2OS cells (Fig. 4A). Obviously, miR-466 overexpression inhibited the proliferation and cell cycle, whereas CCND1 restoration enhanced cell proliferation and cell cycle in 143B and U2OS cells (Fig. 4B-E). Consistently, miR-466 overexpression promoted cell apoptosis, whereas CCND1 overexpression reduced apoptotic cells (Fig. 4F). These results further confirmed that miR-466 exerted tumor-suppressive function through targeting CCND1.