MG63 human osteosarcoma cells (American Type Culture Collection, Rockville, MD USA) were maintained in minimum essential medium (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Biowest, Riverside, MO, USA), 1% non-essential amino acids, and 1% penicillin/streptomycin in a 5% CO2 incubator at 37°C. The expression plasmid CFIm25 and empty vector (pcDNA.3.1) were purchased from Tiangene (Tianjin, China). In the transfection experiments, the cells were cultured at a density of 2×105 in serum-free medium without antibiotics at 60% confluence for 24 h in a 5% CO2 incubator at 37°C, and then transfected with transfection reagent (Lipofectamine 2000; Invitrogen; Thermo Fisher Scientific, Inc.), according to manufacturer's protocol. Following incubation for 6 h, the medium was removed and replaced with normal culture medium for 48 h in a 5% CO2 incubator at 37°C.

The locked nucleic acid-modified oligonucleotide inhibitor (anti-miR-181a) used for miRNA knockdown and the scramble control were purchased from Exiqon, Inc. (Woburn, MA, USA).

Total RNA, including miRNA from tissue samples and cells was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of RNA was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). The mRNA was reverse transcribed using a reverse transcription kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The relative levels of miR-181a were examined using altered stem-loop RT-PCR with specific RT and PCR primers. U6 served as a control. The reverse transcription primers for miR-181a and U6 are as follows: RT primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACTCACCGA-3′, specific primer 5′-GAACATTCAACGCTGTCGGTG-3′ and U6 5′-ATCCAGTGCAGGGTCCGAGGTA-3′. Relative quantification of mRNA expression levels was performed according to the manufacturer's instructions (Bio Rad, Hercules, CA, USA). Detection of the mature form of the miRNAs was performed using an mirVana qRT-PCR miRNA detection kit and primer sets, according to the manufacturer's protocol (Ambion; Thermo Fisher Scientific, Inc.). U6 small nuclear RNA was used as an internal control.

Colony formation analysis was performed as described previously (36). Briefly, when the transfected cells were growing in the logarithmic phase they were trypsinized with 0.05% Trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) and seeded into 6-well plates at a density of 2,000 cells/well. The cells were maintained in an incubator at 37°C for 7 days. On day 8, the colonies were washed with phosphate-buffered saline (PBS), fixed with formalin (10%), and stained with methyl violet, all were purchased from Beyotime Institute of Biotechnology (Haimen, China). The methyl violet dye was then washed with PBS and the number of colonies were counted under a microscope (IX53; Olympus Corporation, Tokyo, Japan). The following calculations were then performed: Colony inhibition rate = [(1 - number of colonies in experimental groups) / control group] × 100%; and colony forming efficiency = 1 colony inhibition rate.

The analysis of proliferation was performed using an MTT assay as described previously (37). Cell viability was determined by MTT assay. Briefly, transfected cells were seeded into 96-well plates at a density of 1.0×105 cells/well and 10 µl MTT (concentration, 5 mg/ml; Sigma Aldrich, St Louis, MO, USA) was added into 100 µl medium at indicated time points. The cells were incubated with MTT for ~4 h at 37°C, followed by removal of MTT and addition of 150 µl DMSO. Following incubation with DMSO for 10 min in the dark, absorbance was measured at 570 nm (A570nm) with a microplate reader (Biorad-168-1000XC; Bio-Rad Laboratories, Hercules, CA, USA).

The cells were seeded into 6-well plate at a density of 3.0×105 cells/well and the cells were transfected when the cell density reached ~80% confluency in the second day. At 48 h following transfection, the cells were lysed using RIPA buffer for 30 min at 4°C. The protein concentration was measured by the BCA method. Western blot analysis was performed as described previously (38). Briefly, following incubation with primary antibody anti-BCL2 (cat. no. ab32124; 1:500; Abcam, Cambridge, MA, USA), anti-myeloid cell leukemia 1 (MCL1) (cat. no. ab32087; 1:500; Abcam, Cambridge, MA, USA), anti-c-myc (cat. no. ab32072; 1:500; Abcam), anti-p53 (cat. no. ab1431; 1:500; Abcam), anti-Ki-67 (cat. no. ab16667; 1:500; Abcam), anti-proliferating cell nuclear antigen (PCNA; cat. no. ab1431; 1:500; Abcam), anti-cyclin D1 (cat. no. EPR2241; 1:500; Abcam) and anti-β-actin (cat. no. ab5694; 1:500; Abcam) overnight at 4°C, incubation with IRDyeTM-800 conjugated anti-rabbit secondary antibodies (caat. no. 925-32211; Li-COR Biosciences, Lincoln, NE, USA) was performed for 30 min at room temperature. The specific proteins were visualized using an Odyssey^™ infrared imaging system (Gene Company, Ltd., Lincoln, NE, USA).

The analysis of potential miR target sites was performed using the commonly used prediction algorithm, miRanda (http://www.microrna.org/microrna/home.do).

For the immunofluorescence analysis of MG63 human osteosarcoma cells. The cells were plated on glass coverslips in 6-well plates at density of 3.0×105 cells/well and transfected with 30 nM anti-miR-181a or the scramble control. At 36 h post-transfection, the coverslips were stained with anti-CFIm25 antibodies. Alexa Fluor 488 goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody were used as secondary antibodies (Invitrogen; Thermo Fisher Scientific, Inc.). The coverslips were counterstained with DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for the visualization of nuclei. Microscopic analysis was performed with a confocal laser-scanning microscope (Leica Microsystems, Bensheim, Germany). Fluorescence intensities were measured in a number of areas with 200–300 cells per coverslip, and analyzed using ImageJ 1.37v software (http://rsb.info.nih.gov/ij/index.html).

Total RNA was isolated from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized from the total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and random hexamer primers (Sangon Biotech, Shanghai, China). The thermal cycle profile was as follows: Denaturation for 30 sec at 95°C, annealing for 45 sec at 52–58°C depending on the primers used, and extension for 45 sec at 72°C. The PCR products were visualized on 2% agarose gels stained with ethidium bromide under UV transillumination. RT-qPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The primer sequences for CFIm25 were: Forward (F) 5′-AGTGTTTACAGGCACAAGAGG-3′ and reverse (R) 5′-TTCAGGAACAGGCAAGGA-3. GAPDH primers were: F 5′-GAAGGTCGGAGTCAACGGATTTG-3′ and R 5′-ATGGCATGGACTGTGGTCATGAG-3′. The products were then separated by electrophoresis using 1.5% agarose gels. The bands were visualized using the BioSpectrum 410 multispectral imaging system with a Chemi HR camera 410 (UVP, Upland, CA, USA) and quantified as previosly described (39).

For the analysis of apoptosis, TUNEL staining was performed as described previously (40). The TUNEL assays were performed with the TMR red In Situ Cell Death Detection kit (supplemented with 0.1% sodium citrate; Hoffmann La Roche Inc., Nutley, NJ, USA), according to the manufacturer's instructions.

Data are presented as the mean ± standard error of the mean. Student's t-test (two-tailed) was used to compare between the two groups. Statistical analysis was performed using SPSS version 17 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to identify and compare the expression levels of miR-181a between osteosarcoma tissues and adjacent normal tissues, RT-qPCR was performed on the cancer tissues and normal tissues. The RT-qPCR analysis revealed that the expression of miR-181a in six osteosarcoma tissue samples were significantly higher, compared with those of the adjacent normal tissues (Fig. 1A). These data suggested that miR-181a is an oncogene in osteosarcoma.

To further identify the role of miR-181a in osteosarcoma cells, the MG63 osteosarcoma cells were transfected with anti-miR-181a. Following stable transfection, the expression of miR-181a was detected using RT-qPCR analysis, and colony formation of the MG63 cells was assessed using a colony formation assay. The results showed that exogenous anti-miR-181a significantly downregulated the expression of miR-181a in the MG63 cells (Fig. 1B) and silencing miR-181a significantly decreased the colony formation rate of the MG63 cells (Fig. 1C). In addition, MTT assays were performed to detect the proliferation of the MG63 cells transfected with anti-miR-181a and the scramble control. The results showed that anti-miR-181a inhibited the proliferation of the MG63 cells following 48 h of transfection, compared with the scramble-transfected cells (Fig. 1D). In subsequent experiments, western blot analysis was used to identify whether the proteins of proliferation-associated markers were also affected by silencing miR-181a in the cells. The results showed that the protein expression levels of c-myc, PCNA and cyclin D1 were downregulated, and the expression of p53 was upregulated by anti-miR-181a in the cells (Fig. 1E).

On demonstrating that silencing miR-181a inhibited the proliferation of the MG63 cells, to provide further evidence that miR-181a was involved in the regulation of MG63 cell apoptosis, a TUNEL assay was performed to analyze whether silencing miR-181a affected apoptosis in the MG63 cells. The TUNEL assay revealed a change in the apoptotic rate of the MG63 cells transfected with anti-miR-181a. Specifically, silencing miR-181a promoted apoptosis in the MG63 cells (Fig. 1F).

Western blot analysis was also performed to identify whether proteins of apoptosis-associated markers were also affected by miR-181a in the cells. Bcl-2 and MCL1 are important anti-apoptotic molecules (41,42). The results showed that the expression levels of BCL2 and MCL1 were downregulated by anti-miR-181a in the cells (Fig. 1E).

miRNAs/miRs are a class of non-coding RNAs, which are able to regulate gene expression at the post-transcriptional level by binding to the 3′UTR of target mRNAs through partial sequence homology; this inhibits translation and/or mRNA degradation (24–26). The upregulation of specific miRNAs can contribute to the downregulation of tumor suppressive genes (31,43,44). Thus, the present study hypothesized that miR-181a affected proliferation and apoptosis by regulating target genes.

In the present study, targeted genes of miR-181a were screened using TargetScan (http://www.microrna.org/microrna/home.do), and a large number of target genes were identified. The gene of interest, CFIm25, was used as it has been regarded as a tumor suppressor gene in malignant tumors (22). Target sites on the 3′UTR of CFIm25 are shown in Fig. 2A. Therefore, miR-181a may downregulate the expression of CFIm25 by targeting its 3′UTR in osteosarcoma cells.

Western blot analyses was performed on MG63 cells transfected with anti-miR-181c or scramble. The results confirmed that the protein level of CFIm25 was increased in the cells transfected with anti-miR-181a (Fig. 2B). Consistent with the results of western blot analyses, immunofluorescence analyses demonstrated that the protein level of CFIm25 increased in MG63 cells transfected with anti-miR-181a, compared with scramble-transfected groups (Fig. 2C). To identify whether CFIm25 mRNA was increased by the silencing of miR-181a, RT-qPCR analysis was performed to detect the mRNA expression of CFIm25 in the MG63 cells transfected with anti-miR-181a or scramble. The results of the RT-qPCR analysis demonstrated that silencing of miR-181a had no effect on the mRNA expression of CFIm25 in the MG63 cells (Fig. 2D and E).

The observation that silencing miR-181a inhibited proliferation, promoted apoptosis and upregulated the expression of CFIm25 in the MG63 cells cells, indicated that anti-miR-181a inhibited proliferation and promoted apoptosis via upregulating the expression of CFIm25. Thus, the present study investigated the roles of CFIm25 in osteosarcoma.

To assess the expression of CFIm25 in osteosarcoma, western blot analysis was performed on six pairs of osteosarcoma tissues and matched adjacent normal tissue samples. The expression of CFIm25 was consistently lower in the osteosarcoma tissues, compared with the normal tissues (Fig. 3A). Subsequently, CFIm25-expressing plasmids were used, and the roles of CFIm25 in MG63 cell were investigated. In order to demonstrate that CFIm25-expressing plasmids stably upregulated the protein expression of CFIm25, western blot analysis was performed on MG63 cells transfected with CFIm25-expressing plasmids. The results showed that the protein expression of CFIm25 was significantly increased by the CFIm25-expressing plasmids in the cells (Fig. 3B). To determine the roles of CFIm25 in colony formation ability and proliferation of the MG63 cells, colony formation and MTT assays were performed. The results of the colony formation assay demonstrated that CFIm25 suppressed colony formation in the MG63 cells (Fig. 3C) and, consistent with the colony formation assay, the MTT assay showed that CFIm25 inhibited the proliferation of the cells (Fig. 3D). In subsequent experiment, we performed western blot analysis to identify whether the proteins of proliferation-associated markers were also affected by CFIm25 in the cells. The results of the present study showed that the expression of cyclin D1 was downregulated the expression of p53, were upregulated by CFIm25 in the cells (Fig. 3E).

Having demonstrated that overexpressing CFIm25 inhibited the proliferation in MG63 cells, to provide further evidence that CFIm25 was involved in the regulation of MG63 cell apoptosis, a TUNEL assay was performed to analyze whether overexpressing CFIm25 affected apoptosis in MG63 cells. The TUNEL assay revealed a change in the apoptotic rate of the MG63 cells transfected with CFIm25. Specifically, overexpressing CFIm25 promoted apoptosis in the MG63 cells (Fig. 3F).

Western blot analysis was also performed to identify whether the protein levels of apoptosis-associated markers were also affected by CFIm25 in the cells. The results of the western blot analysis showed that the expression levels of BCL2 and MCL1 were downregulated in the CFIm25-overexpressing cells (Fig. 3E).