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miRNA-218 targets multiple oncogenes and is a therapeutic target for osteosarcoma

Survivin is overexpressed in various cancers and is correlated with treatment resistance and prognosis. MicroRNAs (miRNAs) directly regulate several target genes and are potential therapeutic agents for various cancers. The present study evaluated multiple gene targets of miR-218, including survivin, in osteosarcoma and compared the anti-tumor effects of miR-218 with those of YM155, an anti-survivin agent. It assessed the expression levels of miR-218 and survivin in osteosarcoma and osteoblast cell lines, as well as the proliferative, migratory and invasive capacities of cells following treatment with miR-218 or YM155. The form of cell death was assessed using fluorescence-activated cell sorting analysis to examine the expression of invasion ability-related genes. Osteosarcoma cell lines were subcutaneously injected into immunodeficient mice; the mice were then treated with miR-218 or YM155 to assess the anti-tumor effects of these agents. The results showed that miR-218 was downregulated, whereas survivin was overexpressed in the osteosarcoma cell line compared with normal osteoblast cells. The expression of survivin was suppressed upon overexpression of miR-218 (miR-218 group) or administration of YM155 (YM155 group), leading to apoptosis and inhibition of osteosarcoma cell proliferation. Invasion and migration abilities were inhibited in the miR-218 group, but not in the YM155 group. In the animal model, both the miR-218 and YM155 groups showed a reduced tumor volume and decreased survivin expression. In osteosarcoma, miR-218 showed a wider range of therapeutic efficacy compared with YM155, suggesting that miR-218 should be evaluated as a treatment target.


Introduction
Osteosarcoma is the most frequent primary bone malignancy affecting the metaphysis of the long bones in children and adolescents. Perioperative chemotherapy combined with surgical treatment has markedly improved five-year disease-free survival and overall survival rates of 59 and 65% (1); however, treatment-resistant and recurrent cases, with metastases and poor prognosis, show a mean survival time of <1 year (2,3). Driver-gene mutations directly involved in tumorigenesis and growth have been discovered in many cancers, leading to the development of molecularly targeted drugs with high efficacy and response rates (4). Although several molecularly targeted drugs for osteosarcoma have been tested in clinical trials, their effectiveness was observed to be insufficient, probably due to the large number of subtypes and non-unified genetic abnormalities of the disease (5,6). Therefore, the mechanisms of tumorigenesis in osteosarcoma must be determined to improve treatment outcomes.
Survivin, a member of the inhibitor of apoptosis protein family, inhibits apoptosis by suppressing caspase activity (7). While it is not expressed in normal tissues, it is overexpressed in almost all types of cancer, including osteosarcoma (8). In addition, patients with high expression of survivin are reported to have a poor prognosis in various cancers (9)(10)(11)(12)(13)(14)(15). The present authors previously reported that increased expression of survivin is a poor prognostic factor in osteosarcoma (16).
YM155 is a molecularly targeted drug developed as an inhibitor of survivin expression, leading to the suppression of cell proliferation and exhibiting anti-tumor effects by improving drug sensitivity (17). YM155 is a potential therapeutic target undergoing clinical testing for lung cancer and melanoma; however, no difference in patient survival was miRNA-218 targets multiple oncogenes and is a therapeutic target for osteosarcoma reported between treatment with docetaxel alone or in combination with YM155 in a phase Ⅱ study of breast cancer (18). Thus, one-to-one drugs have limitations and therapeutic agents that act more broadly to target multiple genes may be needed. Micro (mi)RNAs are small non-coding RNAs of 18-25 nucleotides that regulate gene expression by binding to the 3'-untranslated region of target mRNAs, thereby regulating various processes such as metabolism, immunity and digestion. Additionally, miRNAs are involved in the progression of numerous diseases, including cancer, making them potential therapeutic targets (19,20).
Of the numerous miRNAs that reportedly target survivin (Table I) (21), the present study focused on miR-218, as it targets genes acting on the NF-κB and the Wnt/β-catenin pathways, which play crucial roles in osteosarcoma tumorigenesis (Table II) (22). miR-218 suppresses survivin expression and exerts antitumor effects in glioblastoma and cervical cancer (23,24). In addition, it improves drug sensitivity and induces apoptosis in lung cancer (25). In osteosarcoma, the mitochondrial apoptotic pathway is thought to be regulated by B-cell lymphoma 2 (Bcl-2) family proteins, including the anti-apoptotic proteins Bcl-2 and Bcl-xl and pro-apoptotic proteins Bax, Bak and Bad (26). Although miR-218 is known to arrest the cell cycle through E2F2 as a tumor suppressor (27), little is known about its role in other processes, such as apoptosis. In addition, the regulation of survivin expression by miR-218 in osteosarcoma has not been evaluated in vivo.
A single miRNA simultaneously targets multiple genes and thus, may normalize an entire failed biological network; however, the therapeutic efficacy of miRNAs compared with that of molecularly targeted drugs remains to be elucidated. The present study evaluated the in vitro and in vivo anti-tumor efficacy of miR-218 in osteosarcoma and compared the effects with those of YM155 to provide a foundation for the development of therapeutic strategies.

Materials and methods
Osteosarcoma and normal osteoblast cell lines. The human osteosarcoma cell lines MG63 and HOS were purchased from the Health Science Research Resource Bank of Japan. The normal osteoblast cell line hFOB1.19 was purchased from ATCC. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.) supplemented with 10% fetal bovine serum (Nichirei Biosciences, Inc.), 0.1 mM non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA) and 100 IU/ml penicillin-streptomycin-glutamine (Thermo Fisher Scientific, Inc.) and incubated at 37˚C with 5% CO 2 .
Phosphate-buffered saline (PBS; NC group) was used as a control.
Reverse transcription-quantitative (RT-q) PCR. At 48 h post-treatment (transfection with miRNA-218 or administration of YM155), total RNA was extracted from each cell type (1x10 5 cells) using the RNeasy Mini kit (Qiagen GmbH). The extracted total RNA was reverse transcribed using Prime Script RT Master Mix (Takara Bio Inc.) to prepare complementary DNA (cDNA) at a concentration of 500 ng/µl according to the manufacturer's recommendations. RT-qPCR for miR-218 was performed using Takara Prime Script and TaqMan Probe (Takara Bio, Inc.) according to the manufacturer's instructions. The PCR reactions were carried out with an initial denaturation for 30 sec at 95˚C followed by 40 cycles of 95˚C for 5 sec and 60˚C for 30 sec. RNU44 (Thermo Fisher Scientific, Inc.) was used as an endogenous control. The synthesized cDNA was used to analyze survivin expression levels using SYBR Premix Ex Taq II (Takara Bio, Inc.). The PCR reactions were carried out with an initial denaturation for 30 sec at 95˚C followed by 40 cycles of 95˚C for 5 sec, 55˚C for 10 sec and 72˚C for 30 sec. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to correct the expression levels with the 2 -ΔΔCq method (28). The experiment was repeated three times. The specific primers used were as follows: miR-218 (assay ID 000521; Thermo Fisher Scientific, Inc.); RNU44 (assay ID 001094; Thermo Fisher Scientific, Inc.); survivin-forward, 5'-ACC GCA TCT CTA CAT TCA AG-3'; survivin-reverse, 5'-CAA GTC TGG CTC GTT CTC-3'; GAPDH-forward, 5'-TCA CCA GGG CTG CTT TTA AC-3'; GAPDH-reverse, 5'-TGA CGG TGC CAT GGA ATT TG-3'.
Western blotting. The cells were lysed using Pierce RIPA buffer (Thermo Fisher Scientific, Inc.) containing a protease inhibitor (Roche Applied Science, Basel, Switzerland). After sonication, the cells were centrifuged and the supernatant was collected for protein extraction. The protein concentration was measured using Bio-Rad DC kits (Bio-Rad Laboratories, Inc.). Cell lysates (20 µg of protein) were electroblotted with 4-12% NuPAGE Bis-Tris gel (Thermo Fisher Scientific, Inc.) at 30 mA. The primary antibodies rabbit anti-survivin (cat. no. 2808; 1:1,000), anti-MMP-2 (cat. no. 13132; 1:1,000) and anti-MMP-9 (cat. no. 13667; 1:1,000; all from Cell Signaling Technology, Inc.) were transferred to difluoride membranes by the semi-dry method and blocked at 4˚C for 1 h with Blocking One (Nacalai Tesque, Inc.) to block non-specific binding. Each primary antibody was diluted with Can Get Signal Immunoreaction Enhancer Solution 1 (Cytiva) and incubated with the membrane for 12 h at 4˚C. The anti-GAPDH rabbit polyclonal antibody (cat. no. ab9485; Abcam,) was used as an endogenous control. After primary antibody treatment, the membranes were washed with 0.1% Tween-containing PBS and incubated with secondary horseradish peroxidase-conjugated anti-rabbit IgG antibodies (cat. no. NA934; Cytiva), each diluted in 0.1% Tween-containing PBS at 1:2,000, for 1 h at 25˚C. Chemiluminescence was induced using Chemi-Lumi One Super assay kit (Nacalai Tesque, Inc.) before bioimaging. Data were visualized and photographed using the analyzer LAS-4000 (Fujifilm). Protein expression was semi-quantified using ImageJ software (ver. 1.53; National Institute of Health) and quantified by correcting for the band concentration of GAPDH for each of the visualized proteins.

Water-soluble tetrazolium salts 8 cell proliferation assay.
Post-treatment (transfection with miRNA-218 or administration of YM155), the cells were seeded into 96-well plates and incubated for 24, 48, or 72 h. Next, the Cell Count Reagent SF (Nacalai Tesque, Inc.) was added to the culture medium and incubated at 37˚C for 1 h. Absorbance was measured using Wallac 1420 ARVO MX (PerkinElmer, Inc.).
Fluorescence-activated cell sorting analysis. Following transfection with miRNA-218 or NC-miRNA or administration of YM155 or PBS, the cells were seeded into 6-well plates, incubated for 48 h, treated with trypsin/EDTA and centrifuged at 300 x g for 3 min at 4˚C. The pellet was dissolved in 500 µl of 1X binding buffer, transferred to a fluorescence-activated cell sorting (FACS) tube, Cells were stained with Annexin V-FITC and propidium iodide using Annexin V-FITC Apoptosis Detection kit (cat. no. K101-25, Abcam) according to the manufacturer's protocols. The cells were sorted by FL1-H (Annexin V channel) and FL2-H (PI channel) on a FACSCalibur (BD Biosciences) f low cytometer and analyzed by CellQuest (v. 3.3; BD Biosciences). The apoptotic rate was calculated as the percentage of early and late apoptotic cells.
Animal model. A total of 24 eight-week-old male nude mice (26.4±1.4 g, BALB/cAJcl-nu/nu) were purchased from CLEA Japan, Inc. and kept in an environment of 22˚C and 60% humidity under a 12-h dark/light cycle, with free access to a food and water. Osteosarcoma cells were lysed in 100 µl Matrigel and 5.0x10 6 cells were injected subcutaneously into the mice. Tumor size was calculated as the long diameter x short diameter x height/2 and experiments were begun when the tumor volume was >120 mm 3 . AteloGene containing miRNAs, miR-218, or NC-miRNAs was injected subcutaneously at the recommended concentration of 25 µM, whereas YM155 (3 mg/kg) and PBS were injected intratumorally (n=6 per group). Each treatment was applied three times at 0, 7 and 14 days. Tumor volumes were measured every 2-3 days until tumor resection at 18 days. Animals were sacrificed with intraperitoneal administration of sodium pentobarbital (100 mg/kg). The tumor tissue was subjected to survivin immunostaining according to the manufacturer's protocols (cat. no. 2808, 1:200; Cell Signaling Technology, Inc.) and the immunohistochemistry score was calculated as the area x density (0-15 points), where the area is the percentage of tumor cells per slide (0, 0%; 1, <5%; 2, 5-20%; 3, 21-50%; 4, 51-75%; 5, >75%) and density can be absent, weak, moderate, or strong, ranging from 0-3, respectively. Each slide was observed once by three individuals. The animal study was approved by the Animal Experiment Committee of Nihon University School of Medicine, Japan (approval number AP16M012) and humane endpoints were also in accordance with this approval. Wound healing assay. The osteosarcoma cells were seeded into 24-well plates at 1.  Statistical analysis. Each experiment was performed at least three times. Data are presented as the means ± standard deviation or median and range. Student's t-test and Mann-Whitney U test were used for comparing two groups and one-way ANOVA or mixed repeated measures ANOVA was used for comparing multiple groups where the Bonferroni's correction was used to compare differences between groups. Statistical analysis was performed using GraphPad Prism 5 software (GraphPad, Inc.). P<0.05 was considered to indicate a statistically significant difference.

miR-218 expression is downregulated in osteosarcoma cell lines compared with normal cells.
To analyze the function of miR-218 in osteosarcoma, its expression levels in osteosarcoma and normal osteoblast cells was first assessed using RT-qPCR. miR-218 expression levels were significantly lower in osteosarcoma cell lines, MG63 and HOS, compared with normal osteoblast cells, hFOB (Fig. 1A). After transfection with miR-218, MG63 and HOS cells overexpressed miR-218 (Fig. 1B). Although differences in transfection efficiency between the two osteosarcoma cell lines were observed, the amount of original miR-218 were significantly lower in MG63 cells than in HOS cells. Therefore, MG63 cells were used in subsequent experiments as it was easier to evaluate the effects of miR-218.

miR-218 overexpression and YM155 administration suppresses survivin expression.
The half-maximal inhibitory concentration (IC 50 ) was measured in a water-soluble tetrazolium salts 8 (WST 8) cell proliferation assay 48 h post-treatment with miR-218 or YM155. The IC 50 of miR-218 was 50 nM, whereas that of YM155 was 5 nM; these concentrations were used in further experiments involving MG63 cells (Fig. 2A). The RT-qPCR results showed that survivin transcription levels were decreased in both experimental groups compared with those in the negative control groups (Fig. 2B). Western blot analysis confirmed these results, revealing decreased protein expression levels of survivin in the miR-218 and YM155 groups ( Fig. 2C and D).

miR-218 overexpression and YM155 administration inhibits cell proliferation.
To investigate the effects of miR-218 overexpression and YM155 administration on cell proliferation in osteosarcoma, a WST 8 assay and FACS analysis were performed. The WST 8 assay showed that the miR-218 and YM155 groups had reduced proliferative capacity compared with the control groups 72 h post-treatment (Fig. 3A). Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.), which was used in gene transfer, has been reported as toxic (29) and probably caused a decrease in the cell number in the NC-miR group. For the same reason, the miR-218 group failed to show a 50% suppression in the cell number compared with the NC-miR group 48 h post-treatment, even though the concentration was set to the IC 50 . However, the NC group was not affected by gene transfer and reached the cell number limit 48 h post-treatment. To evaluate apoptosis 48 h post-treatment, Annexin V-PI staining was performed. FACS analysis showed an increase in the rightward migration of each cell in the miR-218 and YM155 groups, indicating early and late apoptosis (Fig. 3B).

miR-218 and YM155 inhibits tumor volume growth.
The effect of miR-218 overexpression on tumorigenesis in osteosarcoma was evaluated in vivo. In nude mice, miR-218 and YM155 significantly inhibited tumor growth compared with that in the NC-miR and NC groups 18 days post-treatment ( Fig. 4A and B). Survivin immunostaining was performed on tumor specimens and both, the miR-218 and YM155 groups, showed decreased immunohistochemistry scores (Fig. 4C).
Overexpression of miR-218, but not YM155, inhibits cell migration and invasion capacity. Effect of the overexpression of miR-218 in osteosarcoma was assessed using wound healing and migration assays. The former assay showed a decrease in the pore area of the miR-218 group and consequently in its migration capacity; in contrast, no significant difference was observed between the YM155 and NC groups (Fig. 5). Consistently, the migration assay showed inhibition of the invasive capacity in the miR-218 group, but no significant difference was observed between the YM155 and NC groups (Fig. 6). The expression level of MMP-2/9, which is involved in the migration and invasion ability of cells was further assessed. In western blotting analysis, MMP-2/9 levels were found to be decreased in the miR-218 group but showed no significant difference between the YM155 and NC groups (Fig. 7).

Discussion
The present study showed that in osteosarcoma, miR-218 overexpression and YM155 treatment both led to apoptosis and inhibited the cell proliferative capacity in vivo and in vitro. However, miR-218 inhibited cell invasion and migration, whereas YM155 did not. There is no direct comparison in the literature between the multi-target miR-218 and single-target molecularly targeted drugs. In osteosarcoma, which shows a non-unified genetic abnormality, it was demonstrated that miR-218 has a broader effect and is more effective than single-target drugs.
Cancer is a disease caused by genetic abnormalities; however, the mechanism of cancer development remains unclear. Mutations in driver genes directly involved in tumorigenesis and growth have been discovered in certain types of cancer (such as breakpoint cluster region-Abelson tyrosine kinase in chronic myeloid leukemia and genetic mutations of epidermal growth factor receptor in lung cancer) (30,31). Molecularly targeted drugs (such as gefitinib used in lung cancer) are highly efficient against cancers caused by driver-gene mutations and increase the median survival of patients (32,33). Clinical trials of various molecularly targeted agents have been conducted in osteosarcoma. Although antitumor activity of one such agent, cabozantinib, has been reported, its long-term action and effect on metastasis remain unclear, probably due to the non-unified genetic abnormality inherent to osteosarcoma (5,34).
Clinical trials with miRNAs for treating malignant mesothelioma and hepatitis C showed positive outcomes (35,36); however, the use of miRNAs for treating osteosarcoma has not been evaluated. The present study focused on survivin, a therapeutic target gene for anti-tumor drugs, which is not expressed in normal tissues but is overexpressed in tumor cells. In osteosarcoma, miR-34a and miR-203 suppress the survivin gene, causing cell cycle arrest in the G 2 -M phase and inducing apoptosis and improving sensitivity to cisplatin (37). However, their effects on the cell invasive and migratory capacity, as well as their in vivo anti-tumor potency are unknown.
miR-218 is a molecular switch that, in addition to its other actions shown in Table II, suppresses survivin and the Wnt/β-catenin pathway. Lu et al (22) and Fernández et al (38) show that lymphoid enhancer-binding factor (lef) 1, β-catenin and T-cell factor (tcf)/lef are involved in survivin expression. Therefore, miR-218 may directly and indirectly regulate survivin, which is highly expressed in osteosarcoma. Bmi-1, a constituent gene of the NF-κB pathway, is associated with the development, extension and prognosis of osteosarcoma. The Bmi-1 antibody, AbBmi-1, suppresses NF-κB and MMP-9 expression and reduces the proliferative and migratory potential of osteosarcoma cells; thus, it may be useful as a novel therapeutic target (39). In addition, epithelial to mesenchymal transition has been extensively studied for the migratory and invasive potential of tumor cells. It has been reported that miR-218 targets and suppresses the expression of roundabout homolog 1 and epidermal growth factor receptor-coamplified and overexpressed protein, thereby decreasing NF-κB transcriptional activity and suppressing epithelial to mesenchymal transition (40). Wnt/β-catenin-mediated complexes with transcription factors, such as tcf and lef, as well as transcriptional coactivators, are intimately involved in osteosarcoma tumorigenesis, resulting in transcriptional activity of multiple downstream target oncogenes (41). In summary, the present study focused on miR-218 because it is closely involved in osteosarcoma tumorigenesis and has many targets in the NF-κB and Wnt/β-catenin pathways, along with affecting the expression of survivin. The present study investigated the underlying cause of the suppression of invasion and migration in the miR-218 group, but not in the YM155 group, and focused on MMPs, which serve a central role in cancer metastasis, particularly MMP-2/9, which are target genes of miR-218. miR-218 and not YM155, suppressed the expression of MMP-2/9, which is in contrast with a previous study suggesting that migration and invasion were suppressed by YM155 treatment (42,43). This discrepancy may be attributed to the considerably higher concentrations of YM155 used in previous studies (42). Typically, the anti-tumor effect is proportional to the concentration and duration of administration of a drug. In clinical practice, high drug concentrations that show anti-tumor potency often cause strong side effects. Additionally, MMCs were not administered to prevent proliferation in previous studies in the assessment of migration capacity (43). Addition of MMCs can eliminate the impact of the proliferative capacity, allowing accurate assessment of migration and invasion capacity (44). The present study revealed a significant difference between the miR-218 and YM155 groups in the IC 50 for the migration and invasion potentials at the same degree of proliferative capacity inhibition migration and invasion ability. Furthermore, MMC administration minimized the effect of the proliferative capacity, demonstrating   further efficacy and potential of miRNA-targeted treatment methods.
One limitation of the present study is that it used only MG63 cells to compare the antitumor effects of miR-218 and YM155. MG63 cells form highly malignant tumors. It has been reported that osteosarcoma cell lines show characteristic labelling profiles, producing extracellular matrices with different compositions and that differences in tumor malignancy exist among different cell lines (45). Among them, MG63 cells show a higher methylation rate of WW domain-containing oxidoreductase and higher proliferative and invasive capacity compared with HOS cells (46). Additionally, MG63 cells showed high efficiency of miR-218 transfection, which was suitable for the comparative study with YM155. The amount of original miR-218 in MG63 cells was significantly lower than that in HOS cells (Fig. 1A) and the expression of miR-218 following transfection was higher in MG63 cells (Fig. 1B). As the purpose of the present study was to investigate the anti-tumor effect of miR-218 overexpression in comparative analysis with YM155 via target genes including survivin, the MG63 cell line was chosen for the present study. However, further studies in other cell lines with varying levels of malignancy might help to elucidate the role of miR-218 in the treatment of osteosarcoma. The second limitation is that the present study was not able to conduct experiments with clinical specimens of osteosarcoma. In the future, it will be necessary to verify the antitumor effects of miR-218 and YM155 in clinical specimens of osteosarcoma.
Overall, miRNAs may have broad clinical applications as therapeutic molecules, as they re-express originally intrinsic genes. Due to the non-unified genetic abnormality inherent to osteosarcoma, the antitumor effect of miR-218, which regulates more genes than molecularly targeted drugs, may be enhanced in osteosarcoma compared with other carcinomas.