A total of three expression profile datasets [GSE16088(16), GSE197158(17) and GSE63631(18)] were selected and downloaded from the GEO database (https://www.ncbi.nlm.nih.gov) for analysis. Differentially expressed genes (DEGs) were analyzed by GEO2R (19). The screening conditions were as follows: Log2-fold change <-1.5 or >1.5, and an adjusted P-value (adj. P) <0.05. The Venn diagram was constructed using Xiantao tool (https://www.xiantaozi.com), a web analysis tool. Data on AEBP1 expression from the Cancer Genome Atlas (TCGA) (https://www.cancer.gov/tcga) were extracted and analyzed using the UALCAN platform (https://ualcan.path.uab.edu) (20). Additionally, the expression of AEBP1 was analyzed using the Cancer Cell Line Encyclopedia (CCLE) (https://sites.broadinstitute.org/ccle).

Two OS cell lines (U2OS and HOS) were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Briefly, the cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37˚C in a humidified atmosphere containing 5% CO₂.

siRNAs targeting the AEBP1 sequence (sense, 5'-GGUGGUGGCUCGUUUCAUC-3' and antisense, 5'-GAUGAAACGAGCCACCACC-3') and non-silencing sequences (sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-GUGACACGUUCGGAGAATT-3') were procured from Guangzhou RiboBio Co., Ltd. Transfection with siRNAs was carried out using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Briefly, 100 pmol siRNA and Lipofectamine 2000 were added in the medium without FBS and antibiotics. Following incubation for 20 min at room temperature, the mixture was added in a 6-well plate and the medium was replaced with complete medium after 4 h. The cells were harvested after 48 h for the further determination.

Total RNA was extracted from OS cell lines (U2OS and HOS) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The extracted RNA was then reversely transcribed into cDNA using the FastKing RT Kit (ref. no. GKR116; Tiangen Biotech Co., Ltd.). Subsequently, RT-qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme Biotech Co., Ltd.) on an Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Inc.; Thermo Fisher Scientific, Inc.). Briefly, after an initial denaturation step at 95˚C for 30 sec, the amplifications were carried out with 40 cycles at a melting temperature of 95˚C for 10 sec and an annealing temperature of 60˚C for 30 sec; followed by a melting curve analysis at 95˚C for 15 sec, 60˚C for 1 min, and 95˚C for 15 sec. The following primers were used to detect the expression of AEBP1: Forward, 5'-ATGGACTATTACTTTGGGCC-3' and reverse, 5'-GGGTAGTCCTCCTGGTGTCC-3'. β-actin was selected as the reference gene, and its expression was detected using the following primers: Forward, 5'-GCGTGACATTAAGGAGAAGC-3' and reverse, 5'-CCACGTCACACTTCATGATGG-3'. The relative gene expression was determined using the comparative Cq method (21), and each experiment was repeated at least three times.

Cells (U2OS and HOS) were harvested, and total protein was extracted using RIPA buffer supplemented with fresh protease and phosphatase inhibitors (Beyotime Institute of Biotechnology) following the manufacturer's instructions. The protein concentration was determined using a BCA assay (Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of proteins (40 µg) were subjected to SDS-PAGE using 10% gels and transferred onto PVDF membranes. The membranes were blocked with 3% BSA (cat. no. KGL2314-10; Nanjing KeyGen Biotech Co., Ltd.) in 10 mM Tris-HCl (pH 7.4, with 0.05% Tween-20) for 1 h at room temperature and then incubated with primary antibodies at 4˚C for 12 h. After washing with Tris-HCl buffer three times, the membranes were incubated with the corresponding peroxidase-conjugated secondary antibody (Abcam) at room temperature for 1 h. After washing with Tris-HCl buffer three times again, the protein bands were visualized using Super-Signal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.), and their densitometry was quantified using ImageJ v1.8.0.345 software (National Institutes of Health). The detailed information regarding the antibodies is presented in Table SI. Each experiment was repeated at least three times.

OS (U2OS and HOS) cells (5,000 cells per well) were seeded in a 96-well plate in triplicate and cultured in a complete medium at 37˚C for 1-5 days. Following incubation, 100 µl of MTT (cat. no. ST316; Beyotime Insitute of Biotechnology) solution (5 mg/ml) was added to each well, and the plate was further incubated at 37˚C for 4 h. Following incubation, the supernatant was carefully removed, and 150 µl of DMSO was added to each well. The plate was then oscillated for 30 min at room temperature to dissolve the formazan crystals. The absorbance at 490 nm was measured using a microplate reader, and the values were determined after subtracting the background. Each experiment was conducted at least three times.

Briefly, 400 cells (U2OS and HOS) were seeded in each well of a 6-well plate for the colony formation assay. After 2 weeks of incubation, the cells were washed three times with cold PBS at room temperature. Subsequently, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by three washes with PBS. The cells were then stained with crystal violet dye for 20 min at room temperature. After staining, cell colonies were observed and imaged using a digital camera (Canon DS126211; Canon, Inc.). The plates were washed with distilled water before imaging. Colonies were defined as >50 cells and counted manually.

The cell proliferation rate of U2OS and HOS cells was determined using the EdU incorporation assay following the manufacturer's instructions (BeyoClick^™ EdU-555 EdU Kit; cat. no. C0075S; Beyotime Institute of Biotechnology). Images were captured under a fluorescence microscope (Leica Microsystems GmbH) from five randomly selected areas of each group. The EdU incorporation experiments were repeated at least three times to ensure the reliability and reproducibility of the results.

Cells (U2OS and HOS) were harvested, washed twice with cold PBS, and then fixed with cold 70% ethanol overnight at 4˚C. After fixation, the cells were resuspended in PBS and stained with propidium iodide (PI) for 30 min at room temperature or Annexin V-APC (eBioscience, Inc.) for 30 min at room temperature. Subsequently, the stained cells were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences). FlowJo software (v10.0; TreeStar) was used to analyze the data. This experiment was repeated at least three times to ensure the reliability of the results.

Cell apoptosis analysis was conducted using a TUNEL assay kit (cat. no. KGA1407-20; Nanjing KeyGen Biotech Co., Ltd.) following the manufacturer's instructions. Briefly, the cells were fixed using 4% paraformaldehyde for 30 min at room temperature, and then incubated with 0.2% Triton X-100 for 15 min at room temperature. The cells were washed three times using PBS solution for 5 min at room temperature. The cells were then incubated with TUNEL reagent for 30 min at 37˚C. Finally, the nuclei were stained with DAPI (5 µg/ml) for 10 min at room temperature and the cells were mounted using an antifade mounting medium. A total of five fields were then observed under a fluorescence microscope (Leica Microsystems GmbH). This experiment was repeated at least three times to ensure the reliability of the results.

U2OS cells (20,000 cells per well) were seeded in a 6-well plate in triplicate and cultured in complete medium at 37˚C. Following siRNA transfection for 48 h, an AKT pathway activator, 4 µg/ml SC79 (cat. no. S7863; Selleck Chemicals), was added in the medium. The cells were then cultured for 24 h at 37˚C and harvested for further western blot analysis.

Statistical analysis was conducted using IBM SPSS Statistics software version 20 (IBM Corp.). Data were derived from a minimum of three independent experiments and are presented as the mean ± standard deviation. Unpaired Student's t-test was used for two-group comparisons and one-way analysis of variance (ANOVA) analysis was used for comparisons among multiple groups followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

In order to screen the new molecular target for osteosarcoma, three GEO profiles were analyzed. GSE16088 contained human osteosarcoma tissues and normal tissues. GSE197158 contained osteosarcoma cell lines and normal osteoblast cell lines. GSE63631 contained mice osteosarcoma and normal tissues. Following GEO2R analysis and Venn diagram construction, 25 genes were identified to be upregulated in all the three datasets (Fig. 1A). In addition, there were no genes identified to be downregulated in all the three datasets (Fig. 1B). The fold change value is presented in Fig. 1C. There was one gene that was identified to be upregulated, AEBP1, and to the best of our knowledge, no studies on the association between this gene and osteosarcoma have been reported to date. To explore the relationship between AEBP1 expression and tumors, an analysis using datasets from TCGA, available on the UALCAN database was conducted. The TCGA database includes sarcoma data but does not distinctly categorize osteosarcoma from other sarcoma subtypes. The findings revealed a high expression of AEBP1 (ranked 8th among all 33 types of tumors) in sarcoma tissue compared with other tumor tissues (Fig. 1D). Although sarcoma also includes tumors originating from muscle and adipose tissue, the results suggested that AEBP1 may be associated with osteosarcoma. Furthermore, the expression of AEBP1 was analyzed in cancer cell lines using the CCLE database and markedly high expression was identified in bone cancer cell lines (Fig. 1E and F). Based on these findings, two OS cell lines, U2OS and HOS were selected for further research on AEBP1 expression in OS.

To investigate the role of AEBP1 in regulating OS, AEBP1 was silenced by transfecting siRNA targeting AEBP1 into OS cells. Following siRNA transfection, the expression of AEBP1 at the both mRNA and protein levels was significantly decreased in U2OS and HOS cells (Fig. 2A-D).

The effect of AEBP1 silencing on cell proliferation was then examined using MTT, colony formation assays, and EdU assays. The results of MTT assays revealed that silencing of AEBP1 increased the cytotoxicity of OS cells (Fig. 3A and C). Additionally, the formation of cell colonies was significantly inhibited after silencing of AEBP1, with the number of colonies decreasing by 60.5% in U2OS cells and 67.2% in HOS cells (Fig. 3B and D).

The EdU assay further indicated a significant decrease in proliferation following silencing of AEBP1. The number of EdU-positive cells was significantly reduced in AEBP1-silenced cells compared with control cells, with U2OS cells showing a decrease from 38.2 to 22.3% and HOS cells showing a decrease from 38.8 to 25.0% (Fig. 3E-H).

To elucidate how AEBP1 regulated cell growth, FCM was used to analyze the cell cycle. In U2OS cells, the results showed that the percentage of cells in the G₁ phase was 39.2% in the control group and increased to 46.8% in the AEBP1-silenced group. Conversely, the percentage of cells in the S phase decreased from 36.3% in the control group to 27.6% in the AEBP1-silenced group (Fig. 4A and B). These findings indicated that knockdown of AEBP1 induced G₁ phase arrest in U2OS cells. However, a consistent trend was not observed in HOS cells. Following silencing of AEBP1, there was no significant change in the cell cycle of HOS cells (Fig. 4C and D).

To investigate the role of AEBP1 in regulating the proliferation of OS cells, the changes in apoptosis were evaluated, following AEBP1 silencing, using TUNEL and FCM assays. TUNEL staining revealed a significant increase in the percentage of TUNEL-positive cells in both U2OS and HOS cells following silencing of AEBP1. In U2OS cells, the percentage of TUNEL-positive cells increased from 2.2% in control cells to 9.6% in AEBP1-silenced cells (Fig. 5A and B). Similarly, in HOS cells, the percentage of TUNEL-positive cells increased from 3.2% in control cells to 7.4% after silencing of AEBP1 (Fig. 5C and D). Consistent with these findings, the results of FCM showed a significant increase in the apoptotic rate of U2OS cells after silencing of AEBP1 (6.6%) compared with control cells (2.2%) (Fig. 5E and F). Similarly, the apoptotic rate of HOS cells significantly increased after AEBP1 knockdown (6.2%) compared with control cells (2.5%) (Fig. 5G and H). Considering that silencing of AEBP1 promoted apoptosis, the levels of Bcl-2 and Bax, key regulators of apoptosis, were also assessed. The findings revealed downregulation of Bcl-2 and upregulation of Bax following silencing of AEBP1 (Fig. 5I and J).

To elucidate the mechanism underlying AEBP1-induced proliferation and apoptosis, the AKT signaling pathway, a regulator of cell proliferation and apoptosis (22), was investigated in U2OS cells. The results demonstrated that phosphorylated AKT (p-AKT) levels significantly decreased following AEBP1 silencing, while total AKT levels remained unchanged (Fig. 6A and C). Furthermore, since silencing of AEBP1 induced G₁ phase arrest in U2OS cells, the protein expression level of cyclin D1, a key regulator of the G₁ phase of the cell cycle and a downstream factor of the AKT signaling pathway (23), was examined. The results showed a decrease in the protein level of cyclin D1 following silencing of AEBP1 (Fig. 6A and C).

To further validate the involvement of the AKT signaling pathway, rescue experiments were performed by adding an AKT pathway activator (SC79). It was revealed that SC79 significantly rescued the inhibitory effect induced by AEBP1 silencing (Fig. 6B and D). In conclusion, the data indicated that AEBP1 regulated the proliferation of OS cells primarily through the AKT signaling pathway.