Tissue microarray assay (TMA)
For the Tissue Microarray assay (TMA), brain primary tumor tissue microarray slides (Cat# GL2082) were obtained from US Biomax, Inc. (MD, USA). The forma-linfixed, paraffin-embedded sections underwent deparaffinization, rehydration, and heat-induced antigen retrieval using 10 mM citrate buffer at pH 6.0. Subsequently, the sections were blocked with 3% BSA and then incubated with an ITGA3 antibody. Following the blocking of endogenous peroxidase activity, immunohistochemistry targeting ITGA3 was conducted using a Mouse/Rabbit Probe HRP Labeling kit with DAB Brown reagent (BIOTnA Biotech, Kaohsiung, Taiwan), following the manufacturer’s instructions, and counterstained with hematoxylin. Each sample stained with ITGA3 was evaluated for staining intensity and scored as negative (0), weak (1), moderate (2), or strong (3).
Immunohistochemistry staining
We prepared 4 µm sections from formalin-fixed, paraffin-embedded tissue blocks, which were then deparaffinized and rehydrated. Antigens were retrieved by autoclaving at 121°C for 10 minutes in Target Retrieval Solution, pH 9.0. To block endogenous peroxidase activity, we treated the sections with 3% hydrogen peroxide for 5 minutes at room temperature. After washing with Tris buffer, the sections were incubated in a 1:200 dilution of primary antibody for 5 hours at room temperature. Following another wash with Tris-buffer, the sections were incubated with a secondary antibody conjugated with horseradish peroxidase for 30 minutes at room temperature. Subsequently, the slides were treated with 3,3-diaminobenzidine for 5 minutes, followed by Mayer’s hematoxylin counterstaining for 90 seconds, and then mounted. Staining scores were utilized to categorize the staining into two intensity types: low-level expression and high-level expression. Scores reflecting the proportion of positively stained tumor cells were graded as follows: 0 (no positive tumor cells), 1 (<10%), 2 (10–50%), and 3 (>50%). Cutoff values for ITGA3 were determined based on a measurement of heterogeneity using the log-rank test with respect to overall survival.
Cell culture and transfection with ITGA3 siRNA
All cell lines were obtained from the American Type Culture Collection (ATCC) and maintained at 37°C in a 5% CO2 atmosphere. GBM8401 and GB8901 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS). U-87 MG and SVGp12 cells were cultured in modified Eagle’s medium (MEM) supplemented with 10% FBS. G5T cells were cultured in Dulbecco’s MEM (DMEM) medium supplemented with 10% FBS. SVGp12 cells, isolated from normal tissue, were used as a normal control. For siRNA transfection of astrocytoma cells, we utilized DharmaFECTTM Transfection Reagents (Dharmacon, Lafayette, CO, USA) along with human ITGA3 siRNA constructs. Following transfection with siRNA, cells were cultured for two days before further use. ITGA3 protein expression levels were assessed by western blot analysis. GBM8401 and U-87 MG cells underwent siRNA-induced knockdown of ITGA3. After 48 hours of incubation with ITGA3 siRNA (siITGA3 group) or nonsense siRNA (negative group), western blot analysis was conducted to measure and compare ITGA3 protein expression levels between the control/negative control groups and the siRNA group.
MTT assay
A density of 3× 104 cells was suspended in a culture medium containing 10% FBS and seeded into a 6-well plate with 0.5 ml of medium per well. The cells were then subjected to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 24 hours and 48 hours
Cell cycle analysis
U-87 MG and GBM8401 cells were initially plated in 6-well plates at a density of 1x106 cells and cultured overnight in a cell incubator. The following day, the cells were harvested by centrifugation in a 10ml centrifuge tube, and the supernatant was collected. They were then washed twice with PBS and treated with 1x Trypsin. Afterward, they were incubated at 37°C for 1-2 minutes until detachment. Once detached, the cells were collected, transferred to a centrifuge tube, and spun at 2500 rpm for 5 minutes to remove the supernatant. Next, 1 ml of PBS was added to resuspend the cell pellet, followed by centrifugation again at 2500 rpm for five minutes. Subsequently, 500 µl of PBS was added to disperse the cell pellet, and then 500 µl of 70% ethanol was slowly introduced for cell fixation. The cells were then refrigerated overnight. On the following day, the cells were centrifuged at 2500 rpm for 5 minutes to remove the ethanol-containing supernatant. The cells were washed with 1 ml of PBS, and then 5 μl of RNAse A (100 mg/ml) was added to the PBS solution, which was then incubated at 37°C. After a 30-minute incubation period, 20 µl of propidium iodide (2 mg/ml, final concentration 40 µg/ml) was added, and the cells were incubated at 37°C for an additional 15 minutes. Subsequently, the cells were transferred from the centrifuge tube to a Falcon tube, and the sample was loaded onto the flow cytometry machine (Beckman Coulter FC500 and FACSCalibur, BD, USA). Data analysis was performed using WinMDI 2.8 free software (BD, USA).
Migration assay
U-87 MG and GBM8401 cells were first washed with PBS, followed by the addition of 1x Trypsin. They were then incubated at 37°C for 1-2 minutes until the cells detached naturally. Subsequently, fresh DMEM culture medium was used to collect the cells in a 15ml centrifuge tube. A small aliquot (10µl) of the cell suspension was then added to a cell counter to determine the cell count for each cell type in culture-inserts (U87MG: 1×106, GBM8401: 5×105 cells). The culture-inserts were placed in a cell culture incubator overnight. On the following day, the culture-inserts were positioned under a microscope at 0 hours and observed. They were then examined again at 12 and 24 hours to monitor cell migration and proliferation over time.
Invasion assay
Cell invasion assays were conducted in vitro using Transwell chambers (COR3452; CORNING, Corning, NY, USA). Cells were seeded at a density of 5 × 105 per insert, with 2 ml of medium added to the lower chamber of each Transwell. After incubation for 24 hours, cotton swabs were employed to remove the cells remaining on the upper surfaces of the Transwell membranes. Cells that had migrated through the membranes to the bottom of the insert were fixed, stained with 1% crystal violet, and then photographed. The number of cells in six random high-powered fields was counted to quantify the extent of invasion.
Adhesion assay
U-87 MG and GBM8401 cells were first washed with PBS, followed by the addition of 1x Trypsin. They were then placed in a 37°C incubator for 1-2 minutes until the cells detached naturally. Once detached, a fresh DMEM culture medium was used to collect the cells into a 15ml centrifuge tube. A small volume (10µl) of cell suspension was then added to a cell counter to determine the cell count for each cell type in a 24-well plate (3×104 cells/well). The cells were then placed in the cell culture incubator. After incubation, the cells were fixed with 37% formaldehyde for 15 minutes, followed by washing with PBS. Subsequently, the cells were stained with 0.5% crystal violet (Sigma‐Aldrich; Louis, MO, USA). After staining, the excess crystal violet was rinsed off with water, and the cells were observed and photographed under a microscope.
Western blotting
All samples were lysed in 200 μl of lysis buffer. Subsequently, 50 μg of protein per sample was loaded into the wells of a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and subjected to electrophoresis at 50 V for 4 hours. The separated proteins were then transferred to PVDF membranes. After incubation for 1 hour in blocking buffer, the membranes were incubated with primary antibodies [N-cadherin (1:1000; protein tech; 22018-1-AP), E-cadherin (1:1000; protein tech; 20874-1-AP), and β-actin (1:20000; Sigma; A5441)] for 2 hours at room temperature. Subsequently, the membranes were incubated with secondary antibodies (AP132P and AP124P; Millipore, Billerica, MA, USA) or a secondary antibody (IRDye; Li-COR, USA) for 90 minutes. Enhanced chemiluminescence solution (Western Lightning, 205-14621; Perkin Elmer, Waltham, MA, USA), a MiniChemiTM imaging and analysis system (Beijing Sage Creation, Beijing, China), and a near-infrared imaging system (Odyssey LI-COR, USA) were utilized to detect specific protein bands. Data analysis was performed using Odyssey 2.1 software.
Statistical analysis
A one-way analysis of variance (ANOVA) was employed to compare the results of proliferation, migration, and invasion assays. A p-value < 0.05 was deemed significant. All statistical analyses were conducted using SPSS 24.0 software (SPSS, Inc., Chicago, IL, USA).