Mouse anti-human USP1 polyclonal antibody was purchased from the ProteinTech Group (Wuhan, China); mouse anti-human monoclonal antibody to Bcl-2 and mouse anti-human polyclonal antibodies to MMP-2, GSK-3β, Stat3, cyclin E1, Notch1, Wnt-1 and cyclin A1 were purchased from ImmunoWay Biotechnology Co. (Plano, TX, USA); MG132 and mouse anti-human monoclonal antibody to USP1 were obtained from Merck Millipore (Darmstadt, Germany); mouse anti-human monoclonal antibody to SIK2 were obtained from BioLegend (San Diego, CA, USA); TRIzol reagent, Lipofectamine™ 2000, Opti-MEM and the SuperScript III reverse transcriptase (RT) kit were obtained from Invitrogen Corp. (Carlsbad, CA, USA); Taq DNA polymerase was purchased from Fermentas, Inc. (Waltham, MA, USA); cell culture media and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA).

Tissue samples from 30 osteosarcomas were obtained from patients who underwent surgery at the Third Affiliated Hospital of Soochow University. All participants who had undergone surgical resection signed an informed consent form for this study. All osteosarcoma cases had been confirmed both clinically and pathologically. The experimental protocols for the present study were approved by the Hospital’s Protection of Human Subjects Committee.

The human osteosarcoma U2OS cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) in 2014 and were identified by short tandem repeat (STR) method in 2015. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Surgical specimens were fixed in 10% formalin solution and embedded in paraffin. Four-μm thick sections were deparaffinized, rehydrated and immersed in 3% hydrogen peroxide solution for 15 min to block endogenous peroxidase activity. For antigen retrieval, the sections were boiled in EDTA buffer (pH 9.0) for 10 min. Histologic sections were pre-treated in 10% normal goat serum in TBS for 30 min at room temperature in order to block non-specific binding sites. Then sections were incubated with polyclonal antibody against USP1 at a dilution of 1:100 in phosphate-buffered saline (PBS) at 4°C for 1 h. After being rinsed five times with PBS, sections were incubated with goat anti-mouse IgG conjugated with horseradish peroxidase at room temperature for 1 h. Sections were developed with diaminobenzidene (DAB) as a chromogen and counterstained with haematoxylin. The sections were analyzed under light microscopy.

The human USP1-specific small interfering RNA (siRNA) sequence is 5′-GAAAGCTCCACATCAATAA-3′, designed with an online Invitrogen software using the USP1 sequence (GenBank code: NM_003600) as a reference. The non-silencing sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as a scrambled control. The recombinant lentiviral particles were regenerated in 293T cells by co-transfection of the modified pGCSIL-GFP viral vector together with the pHelper 1.0 and pHelper 2.0 plasmids using the Lipofectamine™ 2000 reagent. The lentiviral particles were referred to as LV-USP1 siRNA or USP1-siRNA. We generated lentiviruses that express non-silencing-shRNA as controls and named them scramble siRNA or scr-siRNA. The osteosarcoma cells transfected with the USP1-siRNA or scrambled siRNA were designated USP1-siRNA and scr-siRNA, respectively. For lentiviral transduction, U2OS cells were cultured in 6-well plates at 70% confluence and infected with control lentivirus or USP1-specific siRNA lentivirus at MOI of 20. After 5 days of infection, U2OS cells expressing GFP protein were observed by fluorescence microscopy to determine the infection efficiency.

The siRNA sequences were designed by commercial software (Applied Biosystems/Ambion, Austin, TX, USA). For SIK2, the siRNA sense sequence is 5-CUA CCGAGAAGUACAAAUADTDT-3′ and the antisense sequence is 5′-UAUUUGUACUUCUCGGUAGdTdT-3′. The siRNA sequences employed as negative controls were 5′-GG CCUCAGCUGCGCGACGCdTdT-3′ (sense) and 5′-GCGUC GCGCAGCUGGGCCAdTdT-3′ (antisense). The selected sequences were confirmed by NCBI BLAST (http://www.ncbi.nlm.nih.gov/blast/) to make sure that the selected genes were specifically targeted. Chemically synthesized siRNAs were designed and sequenced by Guangzhou RiboBio, Co., Ltd. (Guangzhou, China).

U2OS cells were transiently transfected with synthetic siRNA using Lipofectamine 2000 reagent according to the manufacturer’s instructions. Briefly, U2OS cells were seeded onto 6-well plates in the maintenance medium at a density of 2×10⁵ cells/well and allowed to grow for 12 h. Then, 5 μl Lipofectamine 2,000 and 200 pmol siRNA were diluted in serum-free medium to a total volume of 250 μl and incubated for 20 min at room temperature. After the cells were washed with DMEM medium without FBS, the diluted siRNA-Lipofectamine mix was added to each well and incubated for 24 h. Then the mix was replaced with fresh DMEM medium containing 10% FBS. Forty-eight hours after the transfection, the U2OS cells were collected for protein and RNA isolation. The osteosarcoma cells transfected with the SIK2-siRNA or scrambled siRNA were designated SIK2-siRNA and scr-siRNA, respectively.

Total RNA was extracted from osteosarcoma cells using TRIzol reagent following the manufacturer’s instructions. RNA samples (1 μg/reaction) were reverse-transcribed with the RevertAid First Strand cDNA Synthesis kit according to the manufacturer’s recommended protocol. The RT reaction was used for amplification with Taq polymerase and a SYBR-Green RT-PCR kit (Takara, Kyoto, Japan) was used to detect specific real-time RT-PCR products. The resulting cDNA was amplified using specific primers, which were designed by using the GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). The primer sequences were: For USP1, sense: 5′-AGGTTG CTAGTACAGCGTTTGC-3′ and antisense: 5′-CACTGGATT CCTTGTTTCTATCAGA-3′; for SIK2, sense: 5′-AGTCTGA AGCCAGGGAAAA-3′ and antisense: 5′-CATGTTGCCAGC AGTTCACC-3′; for β-actin, sense: 5′-GGCACTCTTCCAGC CTTCC-3′ and antisense: 5′-GAGCCGCCGATCCACAC-3′. Relative gene-expression levels were calculated using the comparative Ct method, also called the 2^−ΔΔCT analysis method.

U2OS cells were seeded onto 96-well plates and incubated in corresponding media supplemented with 10% FBS. After 24, 48, 72 and 96 h of incubation, 10 μl of Cell Counting kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was added into each well, followed by 4 h of incubation. The optical density values were measured at 450 nm using a Bio-Rad microplate reader to determine cell viability. The results were based on three independent experiments and are presented as mean ± SD.

Soft agar colony-formation assay was used to determine the effect of USP1 knockdown on the transformation capability of U2OS cells. In brief, U2OS cells were counted and inoculated in 6-well plates at a density of 3,000 cells/well. After being cultured at 37°C in 5% CO₂ in a humidified incubator for 2 weeks, U2OS cells were immobilized by 4% paraformaldehyde and stained using Giemsa dye for 20 min. The cells were rinsed with distilled water. Plates were then scanned and photographed under an inverted microscope. All experiments were performed three times.

The U2OS cells were harvested and washed with ice-cold PBS twice. Cells were then resuspended with 100 μl of Annexin V binding buffer, and incubated with APC-labeled Annexin V at room temperature for 15 min. Fluorescence-activated cell sorting (FACS) analysis for Annexin V staining was performed to determine cell apoptosis. Each experiment was performed in triplicate.

Twenty-four hours after the infection, the invasion ability of U2OS cells was determined using a Transwell chamber. A total of 40 μl Matrigel (10 mg/ml) (BD Biosciences, San Jose, CA, USA) was coated to 48-well plates and polymerized at 37°C for 30 min. U2OS cells were collected into DMEM medium, supplemented with 1% FBS. In brief, a 100 μl cell suspension containing 2.0×10⁵ cells was added to the upper chamber of each insert. For each Transwell chamber, 500 μl of DMEM supplemented with 20% FBS was added in the lower chamber. After 24 h of incubation at 37°C, cells and Matrigel on the upper surface of the well were removed. Cells invaded to the lower chamber were fixed with methanol, stained with 0.5% crystal violet for 20 min and then were counted under the light microscope. In addition, invaded cells were segregated, lysed and quantified at 570 nm using spectrophotometer to evaluate the amount of cells.

The U2OS cell pellets were lysed with protein extraction solution and incubated at 20°C for 20 min. After protein quantification, the samples were separated by electrophoresis in SDS-PAGE and then transferred to a PVDF (polyvinylidene fluoride) membrane. After blocking in Tris-buffered saline Tween-20 (TBST) containing 5% non-fat milk for 1 h at room temperature, membranes were subsequently incubated with primary antibodies against USP1, SIK2, matrix metalloproteinase-2 (MMP-2), glycogen synthase kinase-3β (GSK-3β), B-cell lymphoma 2 (Bcl-2), signal transducer and activator of transcription 3 (Stat3), cyclin E1, Notch homolog 1 (Notch1), Wingless-type protein-1 (Wnt-1) and cyclin A1 at 4°C overnight. After incubation with secondary antibodies (1:1,000; peroxidase-conjugated anti-mouse IgG) for another 2 h, the membranes were washed in TBST buffer and protein were detected using enhanced chemiluminescence. GAPDH and β-actin bands were used as loading controls.

The U2OS cells were cultured in 6-well plates at a density of 4×10⁴ cells/well. After 24 h of incubation, cells were treated with various concentrations of MG132 (2, 5 and 10 μmol/l) and cultured for 0, 4, 8 and 16 h. Level of SIK2 protein was determined using western blot analysis.

Data are reported as the mean ± SEM of three independent experiments. Statistical analysis between comparable groups was performed using a one-way ANOVA by using GraphPad Prism 5.0 software. In each case, P<0.05 was considered statistically significant.

We examined the expression of USP1 in 30 samples from patients with osteosarcoma. Of the 30 patients, 26 (86.67%) were classified as positive expression of USP1. USP1 protein with positive staining was shown in the nuclei and cytoplasm of osteosarcoma tissues (Fig. 1A-a), while rare visible USP1 staining was detected in cartilage tumor tissues (Fig. 1A-b) and normal bone tissues (Fig. 1A-c). The USP1 expression in osteosarcoma tissues was significantly higher than that in cartilage tumor tissues and normal bone tissues.

To further illuminate the relationship between USP1 and osteosarcoma, we constructed a lentivirus-based USP1-specific siRNA vector and a scramble-siRNA vector. The two vectors were infected into U2OS cells for 3 days and >90% of the cells showed a green fluorescence indicating successful infection (Fig. 1B). To confirm whether the USP1-siRNA had silenced the expression of USP1 mRNA and protein, real-time PCR and western blot analysis were performed on lentivirus-infected cells. The results showed that both mRNA (Fig. 1C) and protein (Fig. 1D) expression levels of USP1 were significantly reduced by infection of USP1-siRNA, compared to scr-siRNA. These data suggest that the infection with USP1-siRNA effectively downregulates USP1 expression in U2OS cells.

CCK-8 assay was performed to study the effect of USP1-siRNA on U2OS cell growth. As shown in Fig. 2A, the cell growth of USP1-siRNA groups showed a significant (P<0.01) reduction in cell viability in comparison to the scr-siRNA groups. The result of a colony formation assay showed that the number of colonies of the USP1-siRNA group (2249.33±156.31) was significantly less than that of the scr-siRNA group (2751.00±83.07) in U2OS cells (P<0.01) (Fig. 2B). These results demonstrate that the reduction in USP1 expression decreased the ability of U2OS cells to form colonies. Furthermore, these results suggest that cell proliferation is reduced due to the silencing of the USP1 gene.

To determine whether USP1 depletion induced cell apoptosis, flow cytometry was used after the silencing of USP1. The flow cytometric analysis shows that the percentage of apoptotic U2OS cells was 3.88±0.22% in the scr-siRNA group cells, and the percentage of apoptotic cells increased to 20.57±0.64% in the USP1-siRNA group cells (P<0.01) (Fig. 2C). These data suggest that the depletion of USP1 specifically induces apoptosis of the U2OS cells.

Next, we performed an in vitro invasion assay, the results showed that OD value of 570 nm was 0.27±0.04 in the scr-siRNA group and 0.17±0.01 in the USP1-siRNA group (P<0.01) (Fig. 2D). These results demonstrate that USP1-siRNA could reduce the invasion of U2OS cells.

The protein levels of related molecules were also examined. The results showed that USP1 knockdown downregulated SIK2, MMP-2, GSK-3β, Bcl-2, cyclin E1, Notch1, Stat3, cyclin A1 and Wnt-1 in U2OS cells (Fig. 3A). These results suggest that USP1 downregulation inhibits growth and invasion of U2OS cells through these proteins.

We further examined whether proteasome inhibitor MG132 can increase the level of SIK2 protein in U2OS cells. The result showed that cells treated with MG132 (5 μmol/l) for 4 or 8 h exhibited an elevation of SIK2 protein (Fig. 3B). It suggests that MG132 upregulates SIK2 expression by decreasing its protein degradation, and therefore, inhibition of proteasome function increases SIK2 protein level in U2OS cells.

Real-time PCR and western blot analysis were performed to confirm whether the SIK2 siRNA inhibited the expression of SIK2 mRNA and protein. The results showed that SIK2 mRNA and protein expression in SIK2-siRNA group were significantly inhibited compared to scr-siRNA group as shown in Fig. 4A and B. It indicates that the targeted SIK2 siRNA inhibited significantly the expression of SIK2 gene.

Colony-forming assays were performed to determine the effect of SIK2 deletion on colony formation. The results showed that the number of colonies of the SIK2-siRNA group (1492.00±74.65) was significantly less than that of the scr-siRNA group (1713.00±159.46) in U2OS cells (P<0.01) (Fig. 4C). This clearly demonstrates that the number of the colonies decreased significantly with the SIK2 deletion.

The flow cytometric analysis shows that the percentage of apoptotic U2OS cells was 8.05±0.20% in the scr-siRNA group cells, and the percentage of apoptotic cells increased to 27.09±3.07% in the SIK2-siRNA group cells (P<0.01) (Fig. 4D). It indicates that the percentage of apoptotic cells increased significantly with the SIK2 deletion.

The images of cells stained with crystal violet suggests that knocking down the expression of SIK2 resulted in the decrease of the amount of invaded cells. Additional observation of absorption assay also proved that the migration ability of U2OS cells reduced after knocking down the expression of SIK2. The results showed that OD value of 570 nm was 0.13±0.02 in the SIK2-siRNA group and is significantly less than the reading in the scr-siRNA group (0.19±0.01, P<0.01) (Fig. 4E), all of these data suggest that SIK2 was involved in the invasion of U2OS cells.