Gene expression profiles of 37 OS cases were obtained from the Gene Expression Omnibus (GEO, ncbi.nlm.nih.gov/geo/) database (accession no. GSE 39055). Expression profiling was performed using an Illumina HumanHT-12 WG-DASL V4.0 R2 Expression BeadChip (Illumina, Inc.). All mRNA expression datasets were retrieved from the GEO database. Analysis of differentially expressed mRNAs of tumor tissue was performed using the DESeq2 package in R (version 1.20.0) (29). Samples were stratified based on the mean expression levels of PSKH1. Survival and ggplot2 packages in R were used for survival analysis and plotting.

GSEA was used to identify potential biological pathways and processes associated with PSKH1. mRNA expression data were obtained from ArrayExpress (https://www.ebi.ac.uk/biostudies/arrayexpress) (accession no. E-MEXP-3628), including four normal bone and 14 OS tissue samples. The dataset was extracted from the Molecular Signatures Database on the GSEA website (gsea-msigdb.org/gsea/index.jsp). Next, A weighted enrichment analysis was carried out utilizing the GSEA 2.2.2 software, which involved the selection of 1,000 random combinations (30).

Total SAOS2, HOS, MG63 and U2OS and osteoblastic hFOB1.19 cells or transfected SAOS2, U2OS and MG63 cells RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was dissolved in 0.025 ml nuclease-free water and stored at −80°C. cDNA synthesis was performed using the SuperScript First-Strand Synthesis System (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Thermocycling was performed at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 45 sec. qPCR was then performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. PCR amplification was performed in triplicate. Primers were as follows: PSKH1: Forward, 5′-GCTGTGGGACAAGCAAGG-3′ and reverse, 5′-TGGTGGTTCTGAGGGAGG-3′ and β-actin: Forward, 5′-AATGAGCGGTTCCGTTGC-3′ and reverse, 5′-TCTTCATGGTGCTGGGAG-3′. β-actin was used as an internal reference to calculate the relative expression of PSKH1. Relative gene expression was analyzed using 2-ΔΔCq method (31).

The SAOS2, HOS, MG63 and U2OS and osteoblastic hFOB1.19 cells or transfected SAOS2, U2OS and MG63 cells lysate was prepared using ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology). BCA Protein Assay kit was used to determine the concentration of protein. Whole cell lysate (25 µg/lane) was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel and semi-dry transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% skimmed milk in Tris-buffered saline with 0.5% Tween-20 (TBST) for 1 h at room temperature, then incubated overnight at 4°C with primary antibodies in TBST containing 5% skimmed milk. Primary antibodies were as follows: PSKH1 (1:500, cat. no. Sc-514401, Santa Cruz Biotechnology, Inc.), p38 (1:2,000, cat. no. Ab170099, Abcam), phosphorylated (p)-p38 (1:1,000; cat. no. Ab47363, Abcam) and β-actin (1:5,000; cat. no. 66009-1-lg, Proteintech Group, Inc.). Membranes were washed and incubated at room temperature for 30 min with horseradish peroxidase-conjugated with goat anti-rabbit (1:1,000; cat. no. A0208, Shanghai Biyuntian Bio-Technology Co. Ltd.) and anti-mouse secondary antibodies (1:1,000; cat. no. A0216, Shanghai Biyuntian Bio-Technology Co. Ltd.). Membranes were washed three times with TBST and bound proteins were visualized using Immobilon chemiluminescent HRP substrate (Millipore, Sigma) and detected using BioImaging Systems (Tanon Science and Technology Co., Ltd.). β-actin was used as an internal control to verify basal protein expression levels using Image Lab software (version 6.0; Bio-Rad Laboratories, Inc.).

Human OS SAOS2, HOS, MG63 and U2OS and osteoblastic hFOB1.19 cell lines were purchased from the American Type Culture Collection (ATCC). Standard protocols for cell culture were followed (32). SAOS2, HOS and MG63 cells were cultured in a Minimum Essential Medium (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Beijing Solarbio Science & Technology Co., Ltd.). U2OS and hFOB1.19 cells were cultured in RPMI-1640 (Hyclone) supplemented with 10% FBS and 1% penicillin/streptomycin (Beijing Solarbio Science & Technology Co., Ltd.). Cells were incubated at 37°C in a humidified incubator with 5% CO₂. In rescue experiments, cells were pretreated with p38/MAPK inhibitor SB203580 (cat. no. ab120162; Abcam) or DMSO for 1 h at 37°C.

For the knockdown of PSKH1, human short hairpin (sh)RNA sequences (shRNA-1, 5′-CCGGTCCCAGCAGCAAGTCAGTATCTCGAGATACTGACTTGCTGCTGGTGTTTTG-3′, shRNA-2, 5′-CCGGTGCTCTTTGACCGCATCATTCTCGAGAATGATGCGGTCAAAGAGCTTTTTG-3′ and shRNA-3, 5′-CCGGTCCTGAGAATCTGCTCTACTCTCGAGAGTAGAGCAGATTCTCAGGTTTTTG-3′) and negative control (NC) shRNA (5′-CCGGTGTTCAGTGCCTACACGATTCTCGAGAATCGTGTAGGCACTGAACTTTTTG-3′) were synthesized by Genewiz, Inc. at the final concentration of 1 µg/µl and cloned into the PLKO.1 plasmid (Addgene, Inc.) to generate PLKO.1-shPSKH1. The transfection of 293T cells (at 90% confluence) with a combination of PLKO.1-shPSKH1 (1,000 ng), psPAX2 (900 ng), and pMD2G (100 ng; both Addgene, Inc.) was performed utilizing the 2nd generation system and Lipofectamine 2000 (by Invitrogen, Thermo Fisher Scientific, Inc.) following the manufacturer's protocols.293T cells were obtained from ATCC. Transfection was performed for 4 h at 37°C, then DMEM (Gibco) was replaced with complete medium for 72 h at 37°C. The supernatant of 293T cells was collected, centrifuged at 4,000 × g for 10 min at 4°C to remove cell debris, filtered, centrifuged at 7,000 × g for 5 min at 4°C and resuspended in ice-cold PBS to detect the titer. The infectious titer was determined by hole-by-dilution titer assay. The quantity of lentiviral plasmid used for transfection was 5 µg. Cells were divided into PSKH1 knockdown (LvshPSKH1) and NC groups. The SAOS2 and U2OS cells at a density of 5×10⁴ cells/well in a 6-well culture plate were infected with LvshPSKH1 or shNC with a multiplicity of infection of 20. After 48 h transfection at 37°C, the selection of stable cell lines was performed with puromycin (Sigma-Aldrich, Merck KGaA) at 4 µg/ml for 2 weeks. The concentration of puromycin used for maintenance was 1 µg/ml. SAOS2 and U2OS cells were used in further experiments.

In addition, human PSKH1 mRNA sequences were synthesized by Genewiz, Inc. and cloned into the pLVX-Puro vector (Clontech; Takara Bio USA) to generate pLVX-Puro-PSKH1 Plasmids. All constructs were verified by DNA sequencing. A 2nd generation system was used to package the lentivirus. A total of 1,000 ng constructed plasmids together with packaging (100 ng psPAX2) and envelope plasmid (900 ng pMD2G; both Addgene, Inc.) was transfected for 4 h at room temperature using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in 293T cells (90% confluency) according to the manufacturer's instructions. DMEM was replaced with full culture medium for 48 h at 37°C. The supernatant of 293T cells was collected, centrifuged at 4,000 × g for 10 min at 4°C to remove cell debris, filtered, centrifuged at 7,000 × g for 5 min at 4°C and resuspended in ice-cold PBS to detect the titer. The infectious titer was determined by hole-by-dilution titer assay. The quantity of lentiviral plasmid used for transfection was 5 µg. Cells were divided into PSKH1 overexpression (oePSKH1) and vector groups. MG63 cells at a density of 5×10⁴ cells/well in a 6-well culture plate were infected with oePSKH1 with a multiplicity of infection of 5. After 48 h transfection at 37°C,stable cell lines were selected by puromycin (Sigma-Aldrich; Merck KGaA) at 4 µg/ml for 2 weeks. The concentration of puromycin used for maintenance was 1 µg/ml. The cells were used for downstream assay or transplantation.

Cell Counting Kit-8 (SAB Biotech; cat. no. CP002) was used to measure cell proliferation. A total of ~3×10⁴ transfected SAOS2, U2OS and MG63 cells/well was seeded in 96-well plates and cultured overnight at 37°C. A total of ~10 ml CCK-8 solution was mixed with 100 ml medium and added to each well. Samples were incubated for 1 h at 37°C. The optical density was measured at 450 nm using a microplate reader (Perlong Medical Equipment Co., Ltd.).

Stable transfected SAOS2, U2OS and MG63 cells were washed twice with ice-cold PBS and fixed with ice-cold 70% ethanol overnight at 4°C, cells were harvested by centrifugation at 1,000 × g for 5 min at room temperature and resuspended in PBS. Cells were stained with 0.5 ml staining buffer, 25 µl propidium iodide and 10 µl RNase A at 37°C for 30 min in the dark. Cell cycle were detected using FACS Caliber flow cytometer (Beckman Coulter, Inc.) The results were analyzed using FlowJo software (version 10.6.2; BD Biosciences) to determine cell cycle phase.

A wound assay was employed to evaluate cell migration, as previously described (33), Stable transfected SAOS2, U2OS and MG63 cells (5×10⁵/well) were seeded in 12-well plates and grown to nearly 100% confluence in 10% FBS-containing medium, before being washed with PBS and transferred to serum-free medium overnight at 37°C. A 100-microliter pipette tip was used to create an artificial wound through the monolayer. After washing three times with PBS, the wound size was measured after 0, 12 and 24 h. Wound areas were photographed under an inverted fluorescence microscope (XDS-500C, Cai Kang Optical Instrument Co., Ltd., Shanghai, China) with a magnification of ×40. The area of wound closure was quantified using ImageJ software (v1.8.0; National Institutes of Health).

Matrigel (BD Biosciences) and Transwell chambers with 8-µm pores (Corning, Inc.) were used for Transwell invasion assay. Defrosted Matrigel glue at 4°C was diluted in MEM (Hyclone) at a 1:2 ratio. In the upper chamber, 80 µl diluted Matrigel glue was used for the invasion assay at 37°C for 30 min. Transwell chambers were placed in 24-well plates. The lower chamber was filled with 800 µl MEM containing 10% FBS. The upper chamber was filled with 200 µl serum-free medium and 2×10⁴ transfected SAOS2, U2OS and MG63 cells per well were seeded for 24 h at 37°C. A total of 1 ml 0.5% crystal violet solution was added to each well. After 30 min at room temperature, each well was washed three times with 1X PBS. Cells in five randomly selected areas under the light microscope (20× magnification) were counted (CX41RF, Olympus).

Stable transfected SAOS2, U2OS and MG63 cells were seeded in 96-well-plates at 1,000 cells/well and cultivated at 37°C for 21 days until clones (>10 cells) could be seen with the naked eye. Cells were fixed with 100% methanol and incubated for 15 min at room temperature. 0.5% Crystal violet staining was used for 30 min at room temperature to visualize the cells. Each sample was tested in triplicate. The cell clones were counted with the naked eye. The clone formation rate was calculated as follows: (Number of clones/number of inoculated cells) ×100.

A total of 40 nude female BALB/c mice (age, 4–6 weeks; weight, 18–20 g) were obtained from Silaike Experimental Animal Limited Liability Company (Shanghai, China). Mice were maintained in a pathogen-free facility at Tongji Hospital with each cage containing 4–5 mice. Mice were maintained under standard 12/12-h light/dark conditions with food and water ad libitum. They were kept in a controlled environment with regulated temperature (22–24°C) and humidity (65–70%). The present study was approved by the Ethics Committee for Animal Experiments of Tongji Hospital. Animals were handled according to The Guide for the Care and Use of Laboratory Animals (34). All mice were euthanized at the end of the experiment. The total duration of the experiment was 5 weeks. No mice died prematurely during the experiment. Mice were subjected to 4% isoflurane for inhalant anesthesia induction and 1.5% for maintenance. The mice were subcutaneously injected with 5×10⁶ U2OS cells in the armpits of nude mice. A total of 16 mice were randomly distributed into shNC (mice injected subcutaneously with U2OS cells transduced with shNC) and shPSKH1 groups (mice injected subcutaneously with U2OS cells transduced with shPSKH1) (n=8/group). Measurements of tumor length and width were taken weekly. Tumor volume was calculated as follows: Tumor volume=(Length × width²)/2. After 33 days, mice were anesthetized with 4% isoflurane, sacrificed by cervical dislocation and death was confirmed by breathing cessation. Tumors were dissected, weighed and fixed in 10% formalin overnight at 4°C. No animals reached the humane endpoints, as follows: Total tumor volume of 1,500 mm³, 15% loss of body weight, vomiting or inability to stand to reach food and water.

HE staining was performed according to standard protocols (35). The tumor tissue was excised and fixed in 10% formalin for 24 h at 4°C. After alcohol gradient dehydration, ~6-µm-thick coronal sections were embedded in transparent paraffin, and dewaxing was performed with xylene. Each tissue section was incubated at 62°C for 30 min. A series of descending ethanol (100, 90, 80 and 70%) was used to dehydrate samples for 5–10 min and samples were rinsed with distilled water for 10 min. The tissue section was subjected to hematoxylin staining and differentiated using 1% hydrochloric acid alcohol to reduce nonspecific background staining and enhance visual contrast. The tissue was rinsed with water, followed by counterstaining with 5% eosin. The slide was subsequently dehydrated using sequential washes with 80, 95, and 100% alcohol and finally cleared with xylene. The sections were sealed with neutral resin and placed in an oven at 65°C for 15 min. These slides were observed optical microscope (Nikon Corporation) at ×200 magnification.

The tumor tissue was excised and fixed in 10% formalin for 24 h at 4°C. Paraffin-embedded tissue samples were cut into 5-µm-thick sections for IHC. Sections were deparaffinized in xylene and rehydrated through graded alcohol series to water. Sections were rinsed for 10 min in distilled water, and antigen retrieval was performed by autoclaving the sections in 0.01 M citrate buffer (pH 6.0) for 15 min at 121°C. After washing with PBS, non-specific protein binding was prevented by incubating the sample with 5% skimmed milk at room temperature for 30 min. The primary antibody for E Cadherin (cat. no. ab231303; Abcam), Ki-67 (cat. no. ab16667; Abcam) was diluted at 1:100 in a humidified container and incubated overnight at 4°C. Following three washes in PBS, slides were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (dilution1:100, cat. no. A0428) and anti-rabbit IgG secondary antibodies (dilution1:100, cat.no. A0423,both Beyotime Institute of Technology) for 1 h at room temperature. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). All sections were photographed on a Nikon Eclipse fluorescence microscope (Nikon Corporation) at ×400 magnification.

BALB/c nude mice were injected in the tail vein with 1×10⁷ U2OS cells. Animals were randomly distributed into shNC and shPSKH1 groups (n=12/group). All animals were euthanized after 8 weeks. Metastatic nodules in the lungs were counted manually and photographed.

All data are expressed as the mean ± SD. For comparisons between multiple groups, one-way ANOVA followed by Tukey's post hoc test was conducted using GraphPad Prism (GraphPad Prism 8; GraphPad Software, Inc.). Additionally, log-rank test was used to compare Kaplan-Meier survival curves. P<0.05 was considered to indicate a statistically significant difference.

To determine the promotive or suppressive effect of PSKH1 on overall survival of patients, PSKH1 expression was evaluated in OS and normal tissue based on the ArrayExpress database. Expression levels of PSKH1 were significantly higher in OS compared with normal bone tissue (Fig. 1A). PSKH1 expression was negatively associated with overall and recurrence-free survival of patients with OS (Fig. 1B and C). Collectively, these data indicated that high PSKH1 expression independently predicted poor survival for patients with OS.

To understand how PSKH1 contributes to OS development, PSKH1 expression levels were detected in OS cell lines using RT-qPCR and western blotting with human osteoblast hFOB1.19 cells as control. The results showed upregulation of PSKH1 mRNA and protein in OS cells (Fig. 1D and E). These results suggest that PSKH1 may be involved in the development of OS.

MG63 cells with relatively low expression of PSKH1 were infected with PSKH1 lentivirus or control virus and a stable PSKH1 overexpressing cell line was successfully generated (Fig. 2A and B). PSKH1 was successfully knocked down in U2OS and SAOS2 cell lines by infecting cells with lenti-shPSKH1 and lenti-sh-control virus (Fig. 2C-F).

Knockdown of PSKH1 inhibited migration of U2OS and SAOS2 cells (Fig. 3A). The role of PSKH1 in tumorigenesis was investigated using CCK-8 and colony formation assays. Silencing PSKH1 expression significantly decreased proliferation of U2OS and SAOS2 cells (Fig. 4A), whereas cell migration and proliferation were significantly increased in MG63 cells infected with PSKH1 lentivirus compared with NC (Fig. 5A and C). Silencing of PSKH1 by shPSKH1 decreased the colony number of U2OS and SAOS2 cells (Fig. 3B), whereas PSKH1 overexpression yielded the opposite result (Fig. 5B). Together, these results suggested that PSKH1 knockdown inhibited OS cell migration and proliferation.

Transwell assay was performed to verify the effect of PSKH1 on the invasion of OS cells. Silencing PSKH1 expression decreased the number of invaded OS cells (Fig. 4B). However, PSKH1 overexpression showed the opposite effect (Fig. 5D). The effect of PSKH1 on the cell cycle of OS cells was investigated. Compared with the empty vector group, silencing of PSKH1 led to a significant increase in the number of cells in the G1 phase, accompanied by a decrease in the proportion of cells in the S and G2 phase (Fig. 4C). Overexpression of PSKH1 was found to decrease the number of cells in G1 phase, with a corresponding increase in the proportion of cells in the S phase (Fig. 5E). PSKH1 expression levels were positively associated with OS cell proliferation, invasion and cell cycle progression.

The effect of PSKH1 on proliferation of OS cells was assessed using a mouse xenograft model. PSKH1 knockdown notably inhibited tumor growth (Fig. 6A). Immunofluorescence analysis revealed that Ki67 expression was decreased in shPSKH1 group (Fig. 6B). PSKH1 knockdown tumors had significantly smaller volume and weight than control tumors (P<0.01; Fig. 6C), indicating that expression of PSKH1 promoted tumor growth in solid tumors. Overall, these results suggested that PSKH1 knockdown inhibited OS tumor growth in vivo.

A lung metastasis mouse model was also established by injecting shNC- and shPSKH1-U2OS cells into the tail veins of nude mice. Mice were then sacrificed 8 weeks after injection to examine metastatic nodes in the lung. E-cadherin expression was significantly decreased in the shPSKH1 group (Fig. 6D and E). PSKH1 decreased the number of metastatic tumor nodes in mice. Collectively, these results indicated that PSKH1 downregulation inhibited lung metastasis.

The potential mechanisms involved in OS pathogenesis were investigated. GSEA was conducted on OS and normal samples in the ArrayExpress database. PSKH1 expression was significantly associated with the p38 pathway (Fig. 7A). The p38 pathway regulates cell invasion and migration (20). To confirm GSEA results, the association between PSKH1, p38 and p-p38 was investigated in vitro. Overexpression of PSKH1 led to an elevation in the expression of p-p38 and an increase in the p-p38/p38 ratio in MG63 cells (Fig. 7B). Conversely, the silencing of PSKH1 through shPSKH1 resulted in a reduction of the expression of p-p38, resulting in a decrease in the ratio of p-p38/p38 in U2OS and SAOS2 cells (Fig. 7C). The effect of PSKH1 on proliferation, invasion and migration of OS cells via the p38 pathway was investigated in vitro. MG63 cells transfected with PSKH1 overexpression vector were treated with a p38 inhibitor, SB203580. The overexpression of PSKH1 had a noticeable impact in promoting the proliferation and migration of established osteosarcoma (OS) cells. However, the treatment with SB203580 effectively attenuated the migration and proliferation in comparison to the cells in the PSKH1 overexpression group (Fig. 7D and E). Additionally, PSKH1 overexpression also significantly increased the colony formation and invasion of OS cells, as well as the proportion of cells in the S phase. However, the treatment with SB203580 effectively reversed the cell cycle progression, invasion, and proliferation in comparison to the cells in the PSKH1 overexpression group (Fig. 8A-C). Furthermore, SB203580 also effectively restored the ratio of p-p38/p38 in established OS cells, which had been induced by the PSKH1 overexpression (Fig. 8D). Together, these results suggested that PSKH1 served an oncogenic role in the development of OS, potentially by activating the p38 pathway.