The age distribution of patients ranged from 14–22 years, and there were seven patients (all men) >18 years and thirteen patients (all men) <18 years. A total of 20 pairs of OS tissues and adjacent normal tissues (2 cm away from tumor) were collected during surgical resection from the Department of Orthopedics, The Affiliated Wuxi No. 2 People's Hospital of Nanjing Medical University (Wuxi, China) and the Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University (Nanjing, China). All tissues were stored at −80°C until protein extraction or paraffin embedding. The present study was approved by the Ethics Committee of the Affiliated Wuxi No. 2 People's Hospital of Nanjing Medical University (Wuxi, China; approval no. 2019-01-0202-05) and written informed consent was provided by patients or their legal guardians prior to the study start.

The cell culture reagents, including culture medium, fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco; Thermo Fisher Scientific, Inc. The Cell Counting Kit-8 (CCK-8) and EdU Proliferation kits were purchased from Dojindo Molecular Technologies, Inc., and Guangzhou RiboBio Co., Ltd., respectively. The agonist (insulin-like growth factor 1) and inhibitor (LY 294002) of PI3K/AKT pathway were purchased from MedChemExpress, Inc. The primary antibodies used were against PLEK2 (ProteinTech Group, Inc.), N-cadherin (Cell Signaling Technology, Inc.), matrix metalloproteinase (MMP)2 (Cell Signaling Technology, Inc.), vimentin (Cell Signaling Technology, Inc.), PI3K (Cell Signaling Technology, Inc.), phosphorylated (p)-PI3K (Cell Signaling Technology, Inc.), AKT (Cell Signaling Technology, Inc.), p-AKT (Cell Signaling Technology, Inc.) and GAPDH (Jackson ImmunoResearch Labortaories, Inc.).

A total of three Gene Expression Omnibus (GEO) datasets, including GSE12865 (13), GSE14359 (14) and GSE33382 (15), were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo), which compare the mRNA levels of OS tissues with those of normal tissues. After importing the profiles into R studio (version 3.6.0; RStudio, Inc.), the data were normalized and probes were nominated. Differentially expressed genes (DEGs) were identified using R package (Limma; version 3.30.11; http://www.bioconductor.org/). Genes with |log fold change| ≥1 were consdiered DEGs. To investigate the biological processes that PLEK2 participates in, the correlation coefficients between each gene and PLEK2 were calculated to perform gene set enrichment analysis (GSEA).

The OS cell lines, U-2 OS, HOS, Saos-2 and 143B, and human osteoblast hFOB 1.19 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. OS cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin, at 37°C with 5% CO₂. The hFOB 1.19 osteoblast cells were maintained in DMEM/F-12 medium containing 0.3 mg/ml geneticin (Gibco, Thermo Fisher Scientific, Inc.), 10% FBS and 1% penicillin-streptomycin, at 33.5°C with 5% CO₂.

To determine the biofunctions of PLEK2 in OS cells, PLEK2 overexpressing U-2 OS cells and PLEK2 knockdown 143B cells were constructed and verified for PLEK2 expression. Lentiviral vectors carrying human PLEK2 cDNA and short hairpin RNA (shRNA) targeting PLEK2 (sh-PLEK2, 5′-CCTGGAGAATGGGAGTGTTAA-3′) were constructed by Shanghai GenePharma Co., Ltd. When the 2nd cells reached 40% confluence, they were infected with vector (GenePharma Co., Ltd.), PLEK2 (GenePharma Co., Ltd.), shRNA encoding a non-specific control sequence (sh-NC, 5′-TTCTCCGAACGTGTCACGT-3′, MOI=50), or sh-PLEK2 (MOI=50), using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) overnight at 37°C. Stably transfected cell lines were obtained by treatment with 5 µg/ml puromycin (Sigma-Aldrich; Merck KGaA) for 1 week before experimentation. PLEK2 mRNA and protein expression levels in stably transfected cells were validated via reverse transcription-quantitative (RT-q)PCR and western blot analyses, respectively.

The CCK-8 assay was performed to assess OS cell proliferation. Stably transfected cells were seeded into 96-well plates at a density of 1,000 cells/well and allowed to grow for different time periods (every 12 h until 72 h). At the specified time points, 10 µl CCK-8 reagent was added to each well. Following incubation for 2 h, cell proliferation was detected at a wavenlength of at 450 nm, using a spectrophotometer (Thermo Fisher Scientific, Inc.).

To further investigate the effects of PLEK2 on OS cell proliferation, the EdU assay was performed. Cells were seeded into 96-well plates at a density of 4,000 cells/well and allowed to grow for 24 h. Subsequently, 50 µM EdU solution was added to each well and incubated for 2 h. Following fixation with 4% polyformaldehyde, the cells were permeated with 0.5% Triton X-100 and stained with 1× Apollo reagent for 30 min. Nuclei were stained with 1× Hoechst 33342 and cells were observed under a fluorescence microscope (magnification, ×200; Carl Zeiss AG).

Stably transfected OS cells were seeded into 6-well plates at a density of 200 cells/well and allowed to grow for 2 weeks. Following incubation, cell colonies were fixed with 4% polyformaldehyde and visualized by staining with 0.1% crystal violet. The number of cell colonies was counted manually.

To assess the pro-metastatic effects of PLEK2 in OS cells, the Transwell assay was performed using an insert with an 8-µm pore (Corning, Inc.). A total of 10,000 cells were seeded into the upper chambers in 200 µl DMEM with no FBS, and 600 µl DMEM supplemented with 10% FBS was plated in the lower chambers. Following incubation for 24 h, cells were fixed with 4% polyformaldehyde and stained with 0.1% crystal violet for 10 min. Cells that migrated into the lower chambers were observed under a light microscope (magnification, ×200), and the number of cells/field was calculated using ImageJ software (version 2.1.4.7; National Institutes of Health). For the invasion assay, Matrigel was used to pretreat the Transwell inserts at 37°C for 1 h until gel formation occurred, and the migration assay was performed using Matrigel-coated inserts.

Stably transfected cells were seeded into 6-well plates until they reached 100% confluence. A P200 pipette tip was used to scratch the cell monolayers. Cells were subsequently cultured in DMEM medium without serum to exclude the interference of cell proliferation in migration distance. At the indicated time points (0 and 24 h after scratching), the cell layers were observed under a a fluorescence microscope (magnification, ×40), and the migration distance was measured to assess cell motility.

Total RNA was extracted from cells using TRIzol^® reagent (Takara, Japan), following the manufacturer's instructions. The extracted mRNA was subsequently reverse transcribed at 37°C for 15 min and 85°C for 5 sec using the PrimeScript RT Reagent kit (Takara Bio, Inc.). qPCR was subsequently performed (predenaturation at 95°C for 10 min, denaturation at 95°C for 15 sec, renaturation at 60°C for 60 sec and repeat for 40 cycles) using a SYBR Green PCR master mix (Nanjing KeyGen Biotech Co., Ltd.). The following primer sequences were used for qPCR: PLEK2 forward, 5′-GTGCTCAAGGAGGGCTTC-3′ and reverse, 5′-GCTTGTAGTACACCAGCGTGTT-3′; and GAPDH forward, 5′-AATGGGCAGCCGTTAGGAAA-3′ and reverse, 5′-GCGCCCAATACGACCAAATC-3′. PLEK2 expression was normalized to GAPDH and relative expression was calculated using the 2^−ΔΔCq method (16).

Total protein of OS cells or tissues was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing phenylmethylsulfonyl fluoride and phosphatase inhibitor, and protein concentration was determined using the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Following denaturation at 95°C, 30 µg proteins were separated via 10% SDS-PAGE, electrically transferred onto PVDF membranes and blocked with 5% skimmed milk at room temperature for 2 h. The membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Labortaories, Inc., 1:5,000, cat. no. 111-035-003) for 2 h at room temperature. TBST (0.1% Tween-20) was used to wash the membranes between each incubation, and the proteins were visualized using an ECL detection kit (Tanon Science and Technology Co., Ltd.). The expression levels of all proteins were normalized to that of GAPDH and ImageJ software (version 2.1.4.7; National Institutes of Health) was used for densitometry.

For immunohistochemistry analysis, all specimens were embedded in paraffin and cut into 5-µm-thick sections. Following antigen retrieval with citrate sodium and blocking in 5% bovine serum albumin at room temperature for 2 h, the sections were incubated with primary antibody for PLEK2 overnight at 4°C. The sections were incubated with the corresponding HRP-conjugated secondary antibody (Jackson ImmunoResearch Labortaories, Inc., 1:200, cat. no. 111-035-003) for 2 h at room temperature and visualized using 3,3′-diaminobenzidine (Beijing Solarbio Science & Technology Co., Ltd.) for 3 min. The sections were observed under a light microscope (magnification, ×200).

All in vivo animal experiments were approved by the Institutional Animal Care and Use Committee of the Affiliated Wuxi No. 2 People's Hospital of Nanjing Medical University. Mice were housed under a controlled temperature (23±0.5°C), humidity (50±10%) and a 12:12 h light:dark cycle, food, drinking water and litter were changed every 2 days. A total of 16 6-week-old male nude mice (18–20 g) were randomly divided into two groups (sh-NC and sh-PLEK2, n=8) and injected with stably transfected OS cells to perform subcutaneous tumorigenesis analysis. Tumor volumes were examined and calculated (Volume=length × width² × 0.5) every 4 days after injection until mice were euthanized at day 28 post injection. All mice were enthanized by intraperitoneal injection of pentobarbital sodium (100 mg/kg) 4 weeks after injection (17). The death of mice was verified 10 min after injection by loss of movement, breath, heartbeat, corneal reflex and muscular tension. Tumor weight was measured after the tumor node was removed, and Ki-67 expression was detected via immunohistochemistry analysis.

Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). All experiments were performed in triplicate and data are presented as the mean ± standard deviation. Unpaired Student's t-test was used to compare differences between two groups, and paired Student's t-test was used to compare differences between paired OS tissues and normal tissues. Two-way ANOVA followed by Sidak's multiple comparisons test were used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

After analyzing three OS datasets using R language, the results demonstrated that PLEK2 expression was notably higher in OS tissues compared with adjacent normal tissues in all three datasets (Fig. 1A), with log fold change >1 (Fig. 1B). RT-qPCR and western blot analyses were performed to detect PLEK2 expression in OS cell lines and tissues. As presented in Fig. 1C (P=0.0272 for U-2 OS, P=0.0023 for HOS, P=0.0001 for Saos-2 and 143B compared to hFOB 1.19), PLEK2 mRNA expression was significantly upregulated in OS cell lines compared with osteoblast cells, and 143B cells exhibited the highest PLEK2 expression, whereas U-2 OS cells exhibited the lowest expression. Western blot analysis further demonstrated that PLEK2 protein expression was significantly higher in the OS cell lines and tissues (Fig. 1D). In addition, immunohistochemistry analysis demonstrated that PLEK2 was highly expressed in OS tissues (Fig. 1E). Taken together, these results suggest that PLEK2 expression is upregulated in OS.

Given that PLEK2 expression is different in the four OS cell lines assessed in the present study, and 143B cells exhibited the maximum level, whereas U-2 OS exhibited the minimum level, these cell lines were selected to comprehensively investigate the effects of PLEK2 on OS proliferation, based on previous studies (18–22). U-2 OS and 143B cells were transfected with a PLEK2-coding sequence and shRNA targeting PLEK2, respectively, and the mRNA and protein levels of PLEK2 following transfection were detected via RT-qPCR for knockdown (P=0.0002) and overexpression (P=0.0046; Fig. 2A) and western blot analyses (Fig. 2B). The CCK-8, colony formation and EdU assays were subsequently performed. As presented in Fig. 2C, PLEK2 knockdown significantly inhibited OS cell proliferation (P<0.0001), whereas overexpression of PLEK2 promoted OS cell proliferation (P<0.0001). In addition, PLEK2 knockdown suppressed the colony formation of OS cells (P=0.0038), whereas overexpression of PLEK2 increased the number of cell colonies compared with the control group (P=0.0018; Fig. 2D). The results of the EdU assay demonstrated that PLEK2 knockdown decreased the percentage of mitotic cells (P=0.0018), the effects of which were reversed following overexpression of PLEK2 (P=0.0153; Fig. 2E).

A subcutaneous tumor model was established to better understand the role of PLEK2 in OS in vivo. The results demonstrated that PLEK2 knockdown significantly decreased tumor volume and suppressed OS growth (P<0.0001; Fig. 2F and G). Furthermore, the tumor nodes retrieved from the PLEK2-knockdown group were much smaller compared with the control group, with a lower average tumor weight (Fig. 2H and I). Western blotting verified that PLEK2 expression was suppressed in the sh-PLEK2 group (Fig. 2J). Immunohistochemistry analysis demonstrated that PLEK2 knockdown markedly downregulated Ki-67 expression (Fig. 2K).

The Transwell assay was performed to investigate the role of PLEK2 during OS metastasis. The results demonstrated that PLEK2 knockdown suppressed the migratory and invasive abilities of 143B (P=0.0005 for migration and P=0.0018 for invasion), whereas overexpression of PLEK2 promoted cell migration and invasion (P=0.001 for migration and P=0.0016 for invasion, Fig. 3A and B). In addition, the wound healing assay indicated that PLEK2 knockdown decreased the migration distance of 143B cells (P=0.0082), whereas overexpression of PLEK2 enhanced motility of U-2 OS cells (P=0.0007; Fig. 3C).

The EMT process plays a crucial role in tumor metastasis (23); thus, the present study analyzed the expression levels of several EMT-related proteins in OS cells following PLEK2 overexpression or depletion. The results demonstrated that PLEK2 knockdown attenuated the expression levels of MMP-2, N-cadherin and vimentin, the effects of which were reversed following overexpression of PLEK2 (Fig. 3D), which suggests that PLEK2 activates the EMT process.

To investigate the underlying molecular mechanism by which PLEK2 induces the EMT process, single-gene GSEA was performed, which indicated that the PI3K/AKT/mTOR signaling pathway was activated following overexpression of PLEK2 (Fig. 4A and B). Western blot analysis confirmed that PLEK2 knockdown significantly suppressed activation of the PI3K/AKT/mTOR pathway by decreasing the phosphorylation of PI3K and AKT, which resulted in the suppression of EMT. However, the addition of insulin-like growth factor 1, an agonist of the PI3K/AKT pathway (24), was able to reverse the effects of PLEK2 knockdown. In addition, overexpression of PLEK2 activated the PI3K/AKT/mTOR pathway and EMT process, which was reversed by LY 294002, an inhibitor of the PI3K/AKT pathway (25) (Fig. 4C).