2.1 Immunohistochemistry (IHC)
We used a formalin-fixed paraffin-embedded tissue microarray comprising 62 samples from patients with OS and 9 samples from adjacent normal rib bone tissues (Alenabio Biological Technology Company; Xi’an, Shaanxi, China). Clinicopathological data, including age, sex, pathological diagnosis, tumor–node–metastasis grading, and tumor stage, were collected from the medical records of surgically treated patients with OS. No patients had been administered preoperative treatment or had the co-occurrence of other diagnosed tumors. The sample size of patients with T3 stage OS tumor was small (n = 3), and thus, this group was excluded from analysis. Samples for IHC analysis were prepared using a standard method.
2.2 Semiquantitative analysis of immunohistochemical data and bioinformatics analysis
All tissue samples were evaluated by two independent pathologists blinded to the clinical data. A semiquantitative score was generated based on the IHC staining intensity as follows: +, weak staining; ++, moderate staining; +++, intense staining. The R2 platform (http://r2.amc.nl) was used to analyze the public OS dataset, which includes 127 OS samples.
2.3. Cell culture
Human OS cell lines were purchased from ATCC (Manassas, VA, USA), and the human normal osteoblast cell line hFOB1.19 was obtained from Jennio Biotech (Guangzhou, Guangdong, China). All cells were cultured at 37°C in a humidified atmosphere comprising 5% CO2. Foxo1 short interfering RNA (siRNA; sc-35382) and negative control (NC) siRNA were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Cells were transfected with 50 nM Foxo1 siRNA or NC siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.
2.4. Lentivirus infection
Commercially available lentiviral (LV)-LAIR-1 constructs (Tianyucheng Biotechnology, Xi’an, Shaanxi, China) were modified to overexpress LAIR-1. Human OS cells were infected with LV-NC or LV-LAIR-1. The infection efficiency of LV vectors expressing green fluorescent protein (GFP) was evaluated by fluorescence microscopy.
2.5. Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted from cells using TRIzol Reagent (Invitrogen). SuperScript III Reverse Transcriptase (Invitrogen) was used for reverse transcription, and PCR was performed using the SYBR Green Realtime PCR Master Mix (TAKARA, Shiga, Japan). qPCR primers for human genes were purchased from Tsingke Biotech (Beijing, China). Melting curve analysis was performed to verify the specificity of the primers. Relative gene expression was quantified using the comparative Ct method (2−ΔΔCT), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal control.
2.6. Western blotting analysis
Total protein was extracted using a routine procedure and blotted with the following primary antibodies: LAIR-1 (sc-398141; Santa Cruz Biotechnology), phospho-Foxo1 (Ser256) (84192; Cell Signaling Technology, Danvers, MA, USA), Foxo1 (2880; Cell Signaling Technology), phospho-Akt (Ser473) (AF8355; Affinity Biosciences, Cincinnati, OH, USA), Akt (9272; Cell Signaling Technology), proliferating cell nuclear antigen (PCNA; BM0104; Boster Biotech Co., Ltd., Wuhan, China), Twist1 (ab50581; Abcam, Cambridge, UK), Glut1 (NB110-39113, Novus Biologicals, Littleton, CO, USA), and β-actin (30101ES50; Yeasen Biotech Co., Ltd., Shanghai, China).
2.7. Wound healing and transwell migration assays
Cells were seeded into six-well plates, and a scratch was produced in the monolayer after 48 h. Images of the wounded region were captured immediately after the scratch and after 6 and 12 h (T0, T6, and T12, respectively) to monitor cell migration into this region. The percentage of scratch area (% scratch) that showed wound closure was calculated as follows:
% scratch = (width at T0 − width at T6 or T12)/width at T0 × 100
Transwell migration assay was performed using a transwell chamber with 8-μm pores (Millipore, Billerica, MA, USA). Untreated OS cells (blank group) and their corresponding transfectants overexpressing LV-NC and LV-LAIR-1 were seeded (2 × 104 into each well) into the upper chambers in 500 μL of serum-free medium. The lower chambers were filled with complete medium, and all chambers were incubated at 37°C for 24 h. The cells on the upper surface of the membrane were removed, whereas those in the lower chamber were fixed and stained with 0.1% crystal violet. Images were obtained using an inverted microscope (CX41, Olympus, Tokyo, Japan).
2.8. Immunofluorescent staining
Cells were cultured on glass chamber slides. OS cells were fixed, permeabilized, and incubated for 1 h at room temperature with the following with primary antibodies: Glut1 (NB110-39113, Novus Biologicals, Littleton, CO, USA), Twist1 (ab50581; Abcam, Cambridge, UK), and Foxo1 (2880; Cell Signaling Technology). After washing, the cells were incubated with Cy3-labeled goat anti-rabbit secondary antibody and stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics, Basel, Switzerland). Images were obtained using an Olympus microscope. Immunofluorescence intensities were quantified using the ImageJ software (NIH, Bethesda, MD, USA).
2.9. RNA-sequencing (RNA-seq)
Total RNA was extracted from HOS cells transfected with LV-NC (n = 3) and LV-LAIR-1 (n = 3) using an RNeasy Micro kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RNA was quantified using a NanoDrop ND-2000 spectrophotometer (Nanodrop Technologies, Inc., Wilmington, DE, USA). Generation of the transcriptome library and RNA-seq was performed on the BGISEQ platform (BGI Technologies, Shenzhen, China). The Dr. TOM2 system (BGI Technologies ) was used to analyze the transcriptome data.
2.10. Statistical analysis
Data were statistically analyzed using the GraphPad Prism version 6.0 software (GraphPad, Inc., La Jolla, CA, USA); all data are presented as mean ± standard deviation. Data were analyzed using an independent sample t-test for comparisons between two groups. Clinical data were statistically analyzed using the SPSS software (version 10.0; SPSS, Inc., Chicago, IL, USA). Pearson χ2 test was used to evaluate the statistical significance of the association between LAIR-1 expression and clinical data (n = 59). P values of <0.05 were considered statistically significant.