Cell culturing

The U251 human glioblastoma (GBM) cell line was procured from Procell Life Science & Technology (Wuhan, China). The cells were cultivated in Dulbecco’s modified Eagle medium (DMEM, Gibco, Billings, MT, USA) supplemented with 10% fetal bovine serum (FBS) and maintained in a humidified atmosphere with 5% CO2 at 37 °C.

siRNA sequences

The siRNA sequences used in this study were as follows: siBcl-2 sense: 5′-GUACAUCCAUUAUAAGCUGdTdT-3′, siBcl-2 antisense: 5′-CAGCUUAUAAUGGAUGUACdTdT-3′; isiBCL-2 sense: 5′-CCUGUACAUCCAUUAUAAGCUGUGG-3′, isiBCL-2 antisense: 5′-CCACAGCUUAUAAUGGAUGUACAGGGG-3′; siNC sense: 5′-GUGAUUGCGAGACUCUGAdTdT-3′, siNC antisense: 5′-UCAGAGUCUCGCAAUCACGdTdT-3′; and siRIG-I sense: 5′-AGCACUUGUGGACGCUUUAAAdTdT-3′, siRIG-I antisense: 5′-UUUAAAGCGUCCACAAGUGCUdTdT-3′. All of these siRNAs were purchased from Suzhou GenePharma (Shanghai, China).

Establishment of the in vitro assay

Cells were seeded into various types of plates (CORNING, Corning, NY, USA) at the recommended cell density per well and allowed to attach for 48 h. Cells in different groups underwent the following treatments. Cells in the control group received the same conditions as the other groups but without any treatment. In the siNC, siBCL-2, and isiBCL-2 groups, the transfection reagent Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) of a concentration of 100 nM was introduced into different wells for each group. The cells were then incubated in serum Opti-MEM for 4–6 h, followed by medium replacement with a medium containing 10% FBS for continued incubation for 48 h.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

To assess the mRNA levels of Bcl-2, RIG-I, IFN-β, and C-X-C motif chemokine ligand 10 (CXCL10) in cancer cells, qRT-PCR was conducted. U251 cells were subjected to various drug treatments, and total RNA was extracted using a total RNA extraction kit (EZBiosciences, Roseville, MN, USA) following the manufacturer’s protocols. Reverse transcription of RNA into cDNA was performed using a Color Reverse Transcription Kit (EZBiosciences). qRT-PCR was performed using SYBR Green Color qPCR Mix (EZBiosciences) following the manufacturer’s instructions and analyzed using an ABI fluorescence quantitative PCR instrument (QuantStudio 3&5; Thermo Fisher, Waltham, MA, USA). The primers used for PCR amplification were as follows. Bcl-2 sense: 5′-GACTTCTCCCGCCGCTACCG-3′, Bcl-2 antisense: 5’-ACACACACATGACCCCACCGAAC-3’; RIG-I sense: 5′-AGGCAGAGGAAGAGCAAGAGGTAG-3′, RIG-I antisense: 5’-CTTTGGCTTGGGATGTGGTCTACTC-3′; IFN-β sense: 5′-CTTGGATTCCTACAAAGAAGC-3′, IFN-β antisense: 5′-CATCTCATAGATGGTCAATGC-3′; and CXCL10 sense: 5’- CTTCCAAGGATGGACCACACA-3′, CXCL10 antisense: 5′-CCTTCCTACAGGAGTAGTAGCAG-3′. The relative expression of the target genes was normalized to the GAPDH control using the 2−ΔΔCt method.

Cell viability assay

The determination of cell viability was conducted through the cell counting kit 8 (CCK-8) assay (Fude, Hangzhou, China). Briefly, cancer cells were initially seeded in 96-well plates at a density of 3 × 103 cells per well. Following 24 h incubation and 48 h treatment with various drugs, the CCK-8 reagent was introduced into each well and incubated for 1–4 h as per the provided instructions. Subsequently, the optical density values were measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

Colony formation ability

U251 cells were seeded in six-well plates at a density of 2 × 103 cells per well. Following a 24-hour incubation period, the cells were subjected to treatment either without or with a specified amount of drug for 14 days. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.05% crystal violet for 30 min. The number of individual colonies containing at least 50 cells was manually counted under a microscope.

Assessment of cancer cell migration and invasion

For the migration assay, U251 cells were plated in six-well plates at a density of 2 × 105 cells per well. Once the cell growth reached 100% confluence, a sterile 200 µL pipette tip was used to directly create a scratch in the cell layer. The cells were then treated with or without a specified amount of drug, and the distance traveled by the cells between the two boundaries of the scratched area was recorded at 0, 12, 24, 36, and 48 h using phase-contrast microscopy.

For the invasion assay, U251 cells (1 × 104) were placed in the upper chamber of a 24-well Transwell chamber with a polycarbonate membrane (8 μm pore size; Corning). After 4-hour incubation, the cells were treated with or without a specified amount of drug in a serum-free DMEM medium. Following an additional 48 h incubation, the cells from the upper well were gently removed with a cotton swab. Cells that migrated through the filter membrane to the bottom chamber were washed with phosphate-buffered saline (PBS), fixed with methanol for 30 min at 25 °C, and stained with a 0.25% crystal violet solution. The quantification of migrated cells was conducted by counting five randomly selected fields of view per filter under an inverted microscope. For the invasion assay, 100 µL diluted matrix gel was added vertically to the center of the Transwell chamber and incubated at 37 °C for 4–5 h. Subsequently, cancer cells (1.5 × 104) were inoculated into the upper chamber, and the remaining procedures were performed as described in the migration assay.

Determination of apoptosis

To assess isiBCL-2-induced apoptosis in cancer cells, Annexin V/propidium iodide (PI) double staining was conducted. U251 cells were seeded at a density of 2 × 105 cells per well in a six-well plate and subjected to treatment with or without a specific amount of drug. After 48-hour treatment, the cellular status was observed through electron microscopy. Subsequently, cells were harvested and stained with Annexin V/PI using the Annexin V/PI Double Stain Apoptosis Detection Kit (Elabscience, Wuhan, China) following the manufacturer’s protocols. The stained cells were then analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA).

Western blotting analysis

In this study, the following antibodies were employed: BCL-2 (T40056T40056; Abmart, Shanghai, China), BAX (T40051; Abmart), Cytochrome C (T55734T55734; Abmart), PARP (T40050; Abmart), Caspase 3 (T40044; Abmart), Caspase 9 (T40046; Abmart), RIG-I (#20566-1-AP; Proteintech, Wuhan, China), mitochondrial antiviral signaling protein (MAVS, #66911-1-Ig; Proteintech), IRF3 (#11312-1-AP; Proteintech), P-IRF3 (#29528-1-AP; Proteintech), and GAPDH (#60004-1-Ig; Proteintech). Cells were harvested and lysed in radio-immunoprecipitation assay buffer (BL504A; Biosharp, Tallinn, Estonia) supplemented with Halt protease (BS-00-0903; Biosharp) and phosphatase inhibitor cocktail (BL615A; Biosharp) on ice. Subsequently, the cell lysates underwent Western blotting analysis, with GAPDH serving as a loading control.

ELISA

Cytokines in mouse tumor tissue, as well as human IL-6 and IFN-β in cell culture medium, were detected using the Human IFN-β and CXCL10 ELISA Kit (Jiangsu Meiman, China) following the manufacturer’s instructions.

Confocal immunofluorescence microscopy

Following rinsing with PBS, cells were fixed with 4% paraformaldehyde for 30 min, rapidly blocked using QuickBlock™ Blocking Buffer (P0220; Beyotime, Shanghai, China) for 15–20 min, and then incubated with the primary antibody diluted in blocking buffer (1% goat serum in PBS-Tween). The antibodies were subjected to overnight incubation at 4 °C. Subsequently, cells were incubated with a fluorescent secondary antibody, CoraLite488/594 (Proteintech), for 1 h at 25 °C. After staining with the secondary antibody, nuclei were counterstained with DAPI (BS097-10 mg; Biosharp). Fluorescence imaging was carried out using a confocal laser scanning microscope (AX NIS-Elements v5.4; Nikon, Tokyo, Japan), and image processing was conducted using NIS-Elements software (Nikon).

Animal handling

BALB/c nude mice (female, 4–6 weeks old, weighing approximately 20 g) were obtained from Sun Yat-sen University Laboratory Animal Center (Guangzhou, China) and housed in a sterile environment in accordance with standardized animal care protocols. The experiments were conducted in compliance with national regulations.

Assessment of isiBCL-2 anti-tumor activity in vivo

Human glioma U251 cells (1 × 107) were subcutaneously injected into the right thigh of female nude BALB/c mice (4–6 weeks old, weighing 18–20 g). The mice were randomly divided into three groups, each consisting of five mice: control (PBS solution), siBCL-2 (2.5 nmol/20 g), and isiBCL-2 (2.5 nmol/20 g). Intratumoral injections were administered when the tumors reached approximately 80 mm3. PBS, siBcl-2, and isiBcl-2 were used with the in vivo transfection reagent (EntransterTM-in vivo; Engreen Biosystem, Beijing, China) every three days for 22 days. Subsequently, tumor samples and blood samples were collected from the mice. Tumor size was measured every two days using vernier calipers, and tumor volume was calculated using the formula: tumor volume: Tumor volume (mm3) = 0.5 × length × width2. Tumor samples were processed by cutting to a specific weight and homogenized for qRT-PCR and Western blot analysis. Blood samples were centrifuged, and the upper layer of serum was collected and diluted for ELISA analysis.

Immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Mice were subjected to treatment with PBS, siBcl-2, and isiBcl-2. After a 22-day treatment period, the mice were euthanized, and tumor tissues were excised. The excised tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sliced into 4–8 μm sections, and subjected to IHC staining. Anti-Ki67 (GB121141; Servicebio, Wuhan, China), anti-Bcl-2, anti-RIG-I, and anti-major histocompatibility complex class I (MHC-I) antibodies were applied for incubation at 4 °C for 24 h. Subsequently, tissue sections were incubated with biotin-labeled secondary antibodies for 1 h and stained with 3,3’-diaminobenzidine substrate. After hematoxylin restaining and dehydration, the tissue sections were sealed with coverslips, and images were scanned using a high-capacity digital slide scanner system (3DHISTECH, Budapest, Hungary).

For the TUNEL assay, the dewaxed and hydrated tissue sections underwent incubation with proteinase K solution for 30 min at 37 °C, followed by three washes with PBS. Each section was then incubated with 50 µL TUNEL reaction solution, protected from light, at 37 °C for 2 h. Subsequently, the sections were washed three times with PBS, incubated with DAPI staining solution for 10 min at 37 °C, washed with PBS, and dried. Finally, the sections were sealed with anti-fluorescence quenching sealer, and images were captured using a Nikon confocal microscope (AX NIS-Elements v5.4).

Statistical analysis

Statistical analyses were performed using SPSS v17.0 (SPSS, Chicago, IL, USA). The data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Differences between groups were assessed using Student’s t-tests or two-way analysis of variance, with the significance level established at *p < 0.05.