Reagents

Mammalian shRNA interference lentiviral vector (VB010000, VectorBuilder, Guangzhou, China); Fetal Bovine Serum (FBS, F0193, Merck, Darmstadt, Germany); Penicillin-Streptomycin (TMS-AB2, Merck, Darmstadt, Germany); Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/12F) (12634028, ThermoFisher, Waltham, MA, USA); Minimum Essential Medium (MEM, PM150410, Procell, Wuhan, China); McCoy’s 5 A (Modified) medium (16600082, ThermoFisher, Waltham, MA, USA); Trypsin solution (T4299, Merck, Darmstadt, Germany); 4% Paraformaldehyde Fixative (P0099, Beyotime, Shanghai, China); Crystal Violet (32675, Merck, Darmstadt, Germany); Phosphate Buffer Saline (PBS, 10010001, ThermoFisher, Waltham, MA, USA); Pentasorbital Sodium (P3761, Merck, Darmstadt, Germany); Hematoxylin Eosin (HE) Staining Kit (C0105S, Beyotime, Shanghai, China); 10% Bovine Serum Albumin (BSA, V900933, Merck, Darmstadt, Germany); Diaminobenzidine (DAB, D5905, Merck, Shanghai, China); RIPA Lysis Buffer (P0013B, Beyotime, Shanghai, China); BCA Protein Quantification Kit (P0009, Beyotime, Shanghai, China); Polyacrylamide Gel (SDS-PAGE, NP0007, ThermoFisher, Waltham, MA, USA); Polyvinylidene Fluoride (PVDF) Membrane (FFP70, Beyotime, Shanghai, China); Tris Buffered Saline with Tween-20 (TBST) Solution (786–1742, G-biosciences, Shanghai, China); BeyoECL Star (P0018S, Beyotime, Shanghai, China); RNA Extraction Kit (RC112-01, Vazyme, Nanjing, China); PrimeScript™ RT reagent Kit (RR037Q, Takara, Kyoto, Japan); Hieff® qPCR SYBR Green Master Mix (11201ES03, yeasen, Shanghai, China).

Clinical experiment

This study included 21 surgical resection samples, comprising 12 non-metastatic OS tissue samples and 9 lung metastatic OS tissue samples, from patients treated at our hospital between February 2021 and August 2023. All patients provided informed consent, and the study was conducted under the supervision of the Ethics Committee of Yingtan People’s Hospital (YT2024101). Quantitative reverse transcription PCR (RT-qPCR) was used to detect the mRNA expression levels of Id-1 and Snail in non-metastatic and lung metastatic OS samples, and the correlation between Id-1 and Snail expression in lung metastatic OS was analyzed.

Cell culture and treatment

Saos-2 (CL0271), MG-63 (CL0217), 143B (CL0441) OS cell lines, and human osteoblast cell line (hFOB 1.19, CL0140) were procured from fenghbio (Changsha, China). OS cell line U2OS (SNL-054) were procured from sunncell (Wuhan, China). The cells were cultured in an appropriate medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Saos-2 and U2OS cells were cultured in McCOY’s 5 A medium. MG-63 and 143B cells were cultured in MEM medium. hFOB 1.19 cells were cultured in DMEM/F12 medium. The cells were incubated in a humidified environment at 37 °C with 5% CO2.

Constructing cells with stable gene transfection

The shRNA targeting Id-1 (sh-Id-1) and the negative control for lentivirus infection (sh-NC) were purchased from Vectorbuilder (Guangzhou, China) (Vector Name: pLV[shRNA]-Neo-U6 > hID1[shRNA#1]). Human osteosarcoma cell line MG-63 was inoculated into culture dishes and lentiviral infection was performed when the cells reached 60% fusion. sh-Id-1 (1, 2, 3#) and sh-NC were transfected into MG-63 cells, and the target sequences are shown in Table 1. After 48 h of infection, stable infected cells were screened by adding neomycin, and the success of gene expression changes was confirmed by RT-qPCR analysis.

Table 1 The target sequences of shRNAsCell scratch assay

Each group of MG-63 was seeded at 2 × 10^4 cells/well in 24-well plates and cultured to confluence. The medium was aspirated, and cells were scratched with a 10 µL pipette tip. Cells were washed with PBS and incubated in a humidified environment at 37 °C with 5% CO2. Scratch images were taken at 0 and 24 h using a phase-contrast microscope with a built-in digital camera. The wound healing rate was calculated using ImageJ software to analyze the scratch area. The wound healing rate was determined by the formula:

$$\begin\:\text\text\text\text\text\:\text\text\text\text\text\text\text &\:\text\text\text\text = \\ & \quad \fraccratch\:area\:-\:Scratch\:area\:at\:24\:hours} \\ & \quad \:\times\:100\end$$

Transwell assay

Cell invasion was measured using the QCM™ Cell Invasion Assay Kit (ECM551, Merck, Shanghai, China) according to the manufacturer’s instructions. Cells from each group were plated in the upper chamber of a membrane coated with polymerized collagen. The medium in the upper chamber contains low-serum concentration. Complete medium was placed in the lower chamber. After incubation at 37 °C for 24 h, cells that had invaded to the lower surface of the membrane were extracted and counted using a standard microplate reader (at 560 nm). Data are presented as the percentage of invasion relative to the control group.

Animal experiments

Thirty-six Specific Pathogen-Free (SPF) 6-week-old male BALB/c nude mice (17–21 g) were purchased from Hunan Slake Kingda Laboratory Animal Co., Ltd. (Production License No.: SCXK [Hunan] 2016-0002). Mice were acclimatized for seven days in a suitable environment (18–26 °C temperature, 40–70% relative humidity, 12-hour light/dark cycle, free access to food and water) before the experiment. This study strictly adhered to the 3R principle and was approved by the Animal Committee of Yingtan People’s Hospital (Ethics No.YT202413).

Eighteen mice were randomized into three groups (n = 6): Control, sh-NC, and sh-Id-1. The tumorigenic ability of OS cells was analyzed by constructing a subcutaneous tumor model. The stable infected cell lines from each group were resuspended in PBS, and the cell concentration was adjusted to 1 × 10⁷ cells/mL. The cell suspension (100 µL per mouse) was then subcutaneously injected into the mice. Mice were weighed once a week, and tumor volume was measured by calculating the long (L) and short (W) diameters. Tumor volume ( V = \(\:\frac\times\:}^}\)). When the tumor volume in the Control group reached 1000 mm^3, at week four, All mice were euthanized by intraperitoneal injection of pentobarbital sodium (150 mg/kg)., and collecting tumor tissue.

Eighteen mice were randomized into three groups (n = 6): Control, sh-NC, and sh-Id-1. Construction of an OS lung metastasis model by injecting OS cells into mouse tibiae. Mice were anesthetized with sodium pentasorbital (30 mg/kg) via intramuscular injection. After the mice lost their pain response, a 1 cm longitudinal incision was made at the proximal end of the right tibia under a surgical microscope (8× magnification). The skin was incised to expose the proximal tibia, and a 1 mL syringe was used to penetrate the bone cavity. A mouse osteosarcoma lung metastasis model was constructed by injecting 5 µL of stably infected cells (2 × 10^6 cells/5 µL) from each group into the tibia of mice. The skin was closed with 5 − 0 surgical sutures. The entire procedure was performed in a sterile environment [16]. The mice were euthanized, and lung tissues were collected in the fifth week. Lung metastasis nodules on the surface were counted visually.

HE staining

Mouse tissues were fixed in 10% formalin for 24 h. The fixed tissue samples were then dehydrated and cleared through graded ethanol and xylene, followed by embedding in paraffin. The paraffin-embedded tissues were sectioned into 5 μm thick slices using a microtome. The paraffin sections were deparaffinized in xylene and rehydrated through graded ethanol to water, then stained with hematoxylin and eosin. After staining, the sections were dehydrated and cleared again using graded ethanol and xylene. Finally, it was observed under a light microscope after sealing the film by resin.

Immunohistochemistry

Paraffin sections of lung tissue from mice with lung metastasis were baked at 55 °C for 30 min in a constant temperature oven, followed by deparaffinization in xylene and a graded ethanol series. Endogenous peroxidase was eliminated by treatment with a 3% hydrogen peroxide solution. The sections were then sealed in 10% BSA for 1 h. Subsequently, the sections were incubated with specific primary antibodies against Id-1 (1:100, ab230679, abcam, Cambridge, England), Snail (1:100, A5243, ABclonal, Wuhan, China), E-cadherin (1:100, A3044, ABclonal, Wuhan, China), vimentin (1:1000, A19607, ABclonal, Wuhan, China), and N-cadherin (1:100, A3045, ABclonal, Wuhan, China) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibodies (1:10,000, 31460, ThermoFisher, Waltham, MA, USA) at room temperature for 30 min. DAB was used for chromogenic staining, followed by counterstaining with hematoxylin. Imaging was performed using a digital confocal microscope (STELLARIS 8, Leica, Wetzlar, Germany).

WB

Total protein was extracted from tissue and cells using RIPA lysis buffer. The protein concentration was determined using a BCA protein quantification kit. Subsequently, the protein samples were loaded onto an SDS-PAGE gel for electrophoretic separation. The separated proteins were transferred to a PVDF membrane, which was subsequently blocked with 5% non-fat milk. The membrane was incubated overnight at 4 °C with primary antibodies against Id-1 (1:500, A8432, ABclonal, Wuhan, China), Snail (1:1000, A5243, ABclonal, Wuhan, China), E-cadherin (1:1000, A24874, ABclonal, Wuhan, China), vimentin (1:20,000, A19607, ABclonal, Wuhan, China), N-cadherin (1:1000, A3045, ABclonal, Wuhan, China) and GAPDH (1:50000, A19056, ABclonal, Wuhan, China). This was followed by incubation with HRP-conjugated secondary antibodies at room temperature for 2 h. The membrane was washed with TBST solution for 5 min, and bands were detected using BeyoECL Star for 30 s. Band intensity was analyzed using ImageJ software (V1.8.0.112, NIH, Madison, WI, USA). GAPDH was used as an internal reference for quantitative analysis.

RT-qPCR

Total RNA was extracted from homogenized tissue or cells using an RNA extraction kit according to the manufacturer’s instructions. Suitable RNA was then reverse-transcribed to obtain cDNA, which was subjected to PCR reactions according to the instructions provided. The relative expression levels of target genes were analyzed using the comparative threshold method (2−ΔΔCt method). The design of PCR primers is listed in Table 2. GAPDH was used as an internal reference for quantitative analysis.

Statistical analysis

Statistical analysis was performed using Prism 9 software and data are expressed as mean ± standard deviation. Correlation analysis was performed using Pearson’s correlation analysis. Differences between groups were analyzed by t-test, and comparisons involving three or more groups were analyzed by one-way ANOVA and two-way ANOVA, followed by Tukey’s post hoc test. p-values less than 0.05 were considered statistically significant.