DMEM and RPMI-1640 medium were purchased from HyClone; GE Healthcare Life Sciences, FBS was obtained from Gibco; Thermo Fisher Scientific, Inc. and Lipofectamine^® 2000 from Invitrogen; Thermo Fisher Scientific, Inc. Matrigel was obtained from BD Biosciences, and siRNA-LINC00460 and the scrambled siRNA were synthesized by Guangzhou Ribobio Co., Ltd. The following primary antibodies: Anti-CDK4 (1:1,000; cat. no. 12790S), anti-CDK6 (1:1,000; cat. no. 3136P), anti-vimentin (1:1,000; cat. no. 3878S), anti-E-cadherin (1:1,000; cat. no. 4065), anti-N-cadherin (1:1,000; cat. no. 4061P), anti-cyclin D1 (1:1,000; cat. no. 2978P), anti-Slug (1:1,000; cat. no. 9585S) and anti-GAPDH (1:5,000; cat. no. 2118L) were purchased from Cell Signaling Technology, Inc. Goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. nos. G1215 and G1216) and ECL reagent were obtained from Wuhan Sanying Biotechnology. The propidium iodide (PI) was purchased from Beyotime Institute of Biotechnology, and the Cell Counting Kit (CCK)-8, trypsin, penicillin, streptomycin sulfate and crystal violet were obtained from Beijing Solarbio Science & Technology Co., Ltd. Unless otherwise stated, other reagents were obtained from CoWin Biosciences Co., Ltd.

OS cell lines MG-63 (TCHu124) and U2-OS (SCSP-5030) were obtained from the Shanghai Cell Bank of Chinese Academy of Medical Sciences and cultured in DMEM supplemented with 10%, penicillin (100 U/ml) and streptomycin sulfate (100 µg/ml). All cells were maintained in a humidified incubator at 37°C and 5% CO₂. During the logarithmic growth phase, cells were washed three times in PBS and digested with trypsin to obtain single cell suspensions. The cells were subsequently plated out and cultured in 6-well plates for further experimentation.

OS cells in the logarithmic growth phase were replenished with fresh DMEM 2 h prior to transfection. The siRNA sequences against LINC00460 or the negative control (NC) were designed and synthesized by Guangzhou RiboBio Co., Ltd. siRNA (3 µg/ml) was transfected into 3×10⁴ OS cells at a cell density of 3×10⁴ cells for 6 h at 37°C using Lipofectamine^® 2000 transfection reagent, according to the manufacturer's protocol. Subsequently, the medium was replaced and the cells were cultured in DMEM for 48 h for subsequent experiments. The sequences of the LINC00460 siRNAs were as follows: NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; LINC00460-1, 5′-CCAUCCACUUCAAAGUAUUTT-3′; LINC00460-2, 5′-GCCUCUGAAAUGGUGACAATT-3′; LINC00460-3, 5′-GGGAAAGAAGACGCAUUCUTT-3′ and LINC00460-4, 5′-UCACCUUGACUACUGCUAUTT-3′.

Total RNA was extracted from transfected cells using an UltraPure RNA kit (CoWin Biosciences Co., Ltd.), according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using a HiFiScript cDNA Synthesis kit (CoWin Biosciences Co., Ltd.). The reverse transcription reaction conditions were as follows: Incubation at 42°C for 50 min, followed by incubation at 85°C for 5 min to terminate the reaction. qPCR was performed to assess the silencing effects of siRNA using the UltraSYBR mixture (CoWin Biosciences Co., Ltd.). The following primer pairs were used for the qPCR: LINC00460 forward, 5′-CAGAAATCCTCCAGCCCTGTTA-3′ and reverse, 5′-AAGTGTCTTGGGTCATGAGTCC-3′; and β-actin forward, 5′-CCCGAGCCGTGTTTCCT-3′ and reverse, 5′-GTCCCAGTTGGTGACGATGC-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec; 40 cycles of 95°C for 5 sec, 60°C for 30 sec and one cycle of melting curve at 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and 50°C for 30 sec. Expression levels were quantified using the 2^−ΔΔCq method (22) from three independent experiments and normalized to the internal reference gene β-actin.

Cell viability was determined using a CCK-8 assay. After 24 h of transfection with siRNA, cells were trypsinized and resuspended in DMEM; 100 µl of the cell suspension was seeded into 96-well plates at a density of 1×10³ cells/well. Next, 10 µl CCK-8 reagent was added into each well and cells were incubated in a humidified incubator at 37°C and 5% CO₂ for 1.5 h. Optical density (OD) values were determined by measuring the absorbance at a wavelength of 450 nm using a microplate reader. Cell viability was determined every 24 h.

A total of ~800 MG-63 or U2-OS cells/well were seeded into 6-well culture plates for colony formation assays and incubated in the culture medium for 14 days. Subsequently, cells were fixed with 4% methanol for 20 min and stained with 0.1% crystal violet for 30 min at 37°C. The colonies were visualized and quantified by counting stained colonies containing ≥50 cells.

Cells (1×10⁴ cells/well) were seeded in 6-well plates and cultured until the cells reached 95% confluence. A sterile 200-µl tip was used to generate the wound in the monolayer of cells in each well, which was subsequently washed three times and was cultured with serum-free DMEM for 24 h. Subsequently, the cells were visualized using an Olympus IX71 inverted microscope (Olympus Corporation; magnification, ×40). The area of the wound surface was measured with ImageJ software, version 1.41 (National Institutes of Health). The width of each wound was measured at five random fields for quantification. All analysis was performed relative to the starting wound width (0 h time point).

Cell invasion analysis was performed using a Matrigel-coated Transwell assay. Matrigel was defrosted from −20°C to 4°C in a refrigerator overnight and the experiment was performed on ice at all times, with all consumables pre-cooled. A total volume of 100 µl diluted Matrigel in serum free-cold DMEM was added to the upper chamber of the Transwell, which was subsequently inserted into 24-well plates. After incubating the Transwell at 37°C for 4 h, gelled Matrigel was gently washed with warmed serum-free RPMI-1640 medium. After 24 h of transfection, the cells were trypsinized and re-suspended in serum-free 1640 medium at a density of 1×10⁵ cells/ml; 100 µl of this cell suspension (1×10⁴ cells) was plated in the upper chamber and 600 µl 1640 medium containing 10% FBS was plated in the lower chamber. After 24 h of incubation at 37°C, the non-invasive cells remaining in the upper chamber of the Transwell plate were scraped off with a cotton swab. The invading cells on the lower surface of the chamber were fixed with 4% formaldehyde for 15 min at room temperature and subsequently stained with 0.1% crystal violet for 5 min. After washing with PBS, invasive cells were counted using a light microscope (magnification, ×100).

Cellular migration was also detected by a Transwell assay and followed the same protocol as the Matrigel invasion assay, with the exception that Matrigel was not used to coat the Transwell plates.

After 24 h of transfection, cells were removed from the culture medium and incubated in serum-free DMEM for 24 h. Subsequently, 1×10⁶ cells were trypsinized with EDTA-free-trypsin and harvested by centrifugation (984 × g; 20°C; 5 min). Cells were resuspended with ice-cold PBS and harvested by centrifugation (same as previous). Cells were fixed overnight with 70% ice-cold ethanol and then collected by centrifugation (984 × g; 20°C; 5 min). Cells were resuspended in 100 µl PBS containing 100 µg/ml RNase and incubated for 5 min at room temperature. Subsequently, the cell suspension was added to 400 µl PBS containing 50 µg/ml PI at room temperature for 30 sec. Following the addition of PI, cells were immediately subjected to FACS analysis by a BD FACSCalibur™ flow cytometer (BD Biosciences) and FlowJo software, version 4.5 (Tree Star, Inc.) was used to analyze the percentage of cells in each phase of the cell cycle (G0/G1, S or G2/M).

A 1 mg/ml stock solution of Hoechst 33342 dye (Beyotime Institute of Biotechnology) and a 0.5 mg/ml stock solution of PI (Beyotime Institute of Biotechnology) in H₂O were filter-sterilized. Cells incubated in 24-well plates were washed twice with PBS, then subsequently stained with 10 µg/ml Hoechst 33342 combined with 5 µg/ml PI at 37°C for 15 min, according to the manufacturer's protocol. After washing twice with PBS, the cells were visualized and counted under a fluorescence microscope (magnification, ×100).

The enzymatic activities of matrix metalloproteinase (MMP)-9 were analyzed by gelatin zymography, according to previous studies (23–25). A total of 2.5×10⁶ cells were seeded in 6-cm dishes and transfection was performed as previously described. After 24 h, cells were washed twice with serum-free DMEM prior to being cultured in serum-free DMEM for 24 h. The culture supernatant was collected by centrifugation (984 × g; 20°C; 5 min) and the pellet was discarded. Gelatin solution was prepared by dissolving 500 mg gelatin in 50 ml distilled and deionized H₂O for 2 h at room temperature, and was subsequently placed in a 65°C water bath for 15 min to obtain a clear gelatin stock solution with a concentration of 10 mg/ml. A 10% acrylamide gel was prepared and gelatin stock solution was added to obtain a final gelatin concentration of 0.5 mg/ml. 16 µl of supernatant was loaded into each well and were run for 1.5–2 h until the indicator dye reached the bottom of the gel. The gel was washed four times for 15 min with zymogram renaturing buffer at room temperature, and then the gel was incubated overnight in incubation buffer at 37°C. Following incubation, the gel was stained with 0.25% Coomassie Blue R-250 for 4 h at room temperature, followed by the application of destaining solution (20% methanol, 10% acetic acid, 70% distilled and deionized H₂O) with gentle agitation until areas of enzyme activity appeared as transparent bands against the dark blue background. The mixture was rinsed with H₂O until excess staining solution was removed. PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, Inc.) was used as a marker. The clear bands were visualized using Image Scanner III (GE Healthcare) and the intensity of each band was measured using ImageQuant TL v2003 software (GE Healthcare).

After 48 h of transfection, total protein from cells transfected with siRNA-NC or siRNA-LINC00460 was extracted using RIPA lysis buffer containing protease inhibitors at 4°C. Total protein was quantified using a bicinchoninic acid protein assay and 20 µg protein/lane was separated via SDS-PAGE on a 10% gel. The separated proteins were transferred onto a PVDF membrane and blocked for 1 h at room temperature with 5% non-fat milk. The membranes were incubated with the corresponding primary antibodies overnight at 4°C. Membranes were washed three times with TBS-0.1% Tween 20. Following the primary antibody incubation, membranes were incubated with goat anti-rabbit/mouse HRP-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. Protein bands were visualized using a chemiluminescence kit. GAPDH served as the loading control. The protein bands intensity was analyzed using Image J software, version 1.41 (National Institutes of Health).

Statistical analysis of all results was performed using SPSS version 20.0 (IBM Corp.) software. Each experiment was independently conducted ≥3 times and all data are expressed as the mean ± SD. Significant differences between groups were determined using Student's t-test or one-way ANOVA with Bonferroni correction used as the post hoc test. Kaplan Meier analysis was used for survival analysis, and log-rank test was conducted to determine P-values. The prognostic value of LINC00460 in OS was analyzed using the GEPIA database (26). P<0.05 was considered to indicate a statistically significant difference.

The overall and disease-free survival of patients with low LINC00460 expression in OS was significantly improved compared with patients with high LINC00460 expression (P<0.05; Fig. 1A and B).

To investigate the function of LINC00460 on OS progression, siRNA was used to knockdown gene expression. Four siRNA fragments, siRNA-LINC00460-(1–4), were designed and synthesized to silence LINC00460 in MG-63 and U2-OS cell lines. siRNA-LINC00460-3 demonstrated the highest siRNA transfection efficiency in MG-63 and U2-OS cell lines, providing a knockdown efficiency of >36.6 and 47.1% compared with NC, respectively (P<0.01; Fig. 1C and D). Therefore, siRNA-LINC00460-3 was selected as the siRNA fragment used to target LINC00460 for subsequent experiments. To evaluate the effect of LINC00460 on cell viability, CCK-8 and colony formation assays were conducted to detect the effect of LINC00460 knockdown on the viability of MG-63 and U2-OS cell lines. MG-63 and U2-OS cell lines transfected with siRNA-LINC00460-3 demonstrated significantly reduced cell viability by 48–72 h compared with the NC (P<0.05; Fig. 2A). Similarly, the colony formation assay reported a significant decrease in the number and size of colonies formed in both cell lines following LINC00460 gene silencing compared with the NC group (P<0.01; Fig. 2B). These results suggested that the genetic knockdown of LINC00460 significantly affected OS cell viability and growth.

To further investigate how LINC00460 knockdown affects OS cell proliferation, the effect of LINC00460 on apoptosis was evaluated using Hoechst/PI staining. siRNA-LINC00460- transfected OS cells exhibited significant increases in apoptosis compared with the siRNA-NC group (MG-63, 52.93±8.25% vs. 2.61±0.20%; U2-OS, 12.33±2.26% vs. 2.16±0.11%; P<0.01; Fig. 2C). These data demonstrated that the genetic knockdown of LINC00460 increased apoptosis of OS cells.

To determine which phase of the cell cycle was affected by LINC00460 knockdown, flow cytometric analysis was performed to estimate the distribution of cells at different stages of the cell cycle. The results indicated that siRNA-LINC00460 transfection induced a significant accumulation of cells in the G0/G1 phase of the cell cycle in both MG-63 and U2-OS cell lines compared with the siRNA-NC group (MG-63, 62.1 vs. 32.6%, respectively; U2-OS, 59.2 vs. 38.1%, respectively; P<0.05; Fig. 3A and B). This indicated that siRNA-LINC00460 arrested MG-63 and U2-OS cells in the G0/G1 phase. In addition, the percentage of cells in the G2/M phase significantly decreased in both cell lines compared with the siRNA-NC group (MG-63, 28.4±3.21% vs. 10.6±2.20%, respectively; U2-OS, 31.9±4.37% vs. 16.6±2.78%, respectively; P<0.05; Fig. 3A and B). There were no significant changes observed in the S phase of either cell line. Based on these data, it was hypothesized that siRNA-LINC00460 induced G0/G1 phase arrest and triggered cell apoptosis to reduce cell viability in OS.

To further validate the mechanism underlying the G0/G1 phase cell cycle arrest following siRNA-LINC00460 transfection in OS cells, western blot analysis was performed to examine the changes in the protein expression levels of cyclin D1, CDK4 and CDK6 in MG-63 and U2-OS cell lines (Fig. 3C). Cyclin D1, CDK4 and CDK6 expression levels were significantly decreased following the genetic knockdown of LINC00460 with siRNA in both cell lines compared with the siRNA-NC group (P<0.05; Fig. 3C). These data demonstrated that OS cells were arrested in the G0/G1 phase following transfection with siRNA-LINC00460, which may be caused by the downregulated expression of cyclin D1 and CDK4/CDK6.

Following siRNA-LINC00460 transfection, MG-63 and U2-OS cells demonstrated a significantly reduced migratory ability (0.22±0.05 and 0.28±0.03, respectively) compared with the siRNA-NC groups (0.62±0.03 and 0.67±0.08, respectively; P<0.05; Fig. 4A). Similar results were observed using the Transwell migration assay (P<0.05; Fig. 4B and C), which suggested that the genetic knockdown of LINC00460 significantly decreased the migration of OS cells. In addition, the invasive ability of MG-63 and U2-OS cells was significantly decreased by ~61.4 and ~83.1%, respectively, following transfection with siRNA-LINC00460 compared with their respective siRNA-NCs (Fig. 4B and C). These data indicated that the invasive ability of OS cells transfected with siRNA-LINC00460 was significantly inhibited.

MMPs are a family of zinc- and calcium-dependent endopeptidases that selectively degrade components of the extracellular matrix (27). MMPs serve crucial roles in tumor cell progression; in particular, MMP-9 is a 92-kDa gelatinase that has been implicated in tumor invasion, growth and distant metastasis (28). Provided that gelatin is an important substrate of MMP-9, a gelatin zymography assay was used to analyze MMP activity to further investigate the effect of LINC00460 on the invasive and migratory capacity of OS cells. Weaker gelatinolytic intensity was observed in MG-63 and U2-OS cells transfected with siRNA-LINC00460 compared with their respective siRNA-NC groups (Fig. 4D). These findings indicated that MMP-9 activity was decreased in the siRNA-transfected cells and thus, that the knockdown of LINC00460 suppressed the OS cell invasive and migratory ability which may be caused by the decreased activity of MMP-9.

To further confirm whether the suppression of the EMT process could represent a mechanism behind the siRNA-LINC00460-mediated inhibition of cellular migration and invasion in OS cells, western blot analysis was used to examine the expression of EMT marker proteins. siRNA-LINC00460 transfected MG-63 and U2-OS cells were observed to have significantly increased expression levels of E-cadherin, an epithelial marker, and significantly downregulated expression levels of N-cadherin and vimentin mesenchymal markers compared with their respective siRNA-NC groups (Fig. 5A and B). Furthermore, there was a significant decrease in Slug, a mediator of EMT, in both cell lines transfected with siRNA-LINC00460 compared with siRNA-NC groups (P<0.05; Fig. 5A and B). Taken together, these results suggested that the genetic knockdown of LINC00460 inhibited the migratory and invasive properties of OS cells through suppressing EMT and inhibiting the expression of MMP-9.