1. Cell Culture
HT22 cells, originating from primary mouse hippocampal neurons, are cultured in DMEM medium at 37°C and 5% CO2. The culture medium includes 10% fetal bovine serum, 20 mM HEPES, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine.
2. Oxygen and Glucose Deprivation/reoxygenation (OGD/R) model
The HT22 mouse hippocampal neuronal cell line, obtained from the American Type Culture Collection (ATCC), serves as an in vitro model for ischemic stroke. Initially, HT22 cells are cultured in glucose/serum-free DMEM and exposed to a 30-minute anaerobic environment of 95% N2 and 5% CO2. Subsequently, they are returned to regular culture medium and maintained at 37°C in a humidified incubator with 95% air and 5% CO2. In contrast, the control group HT22 cells are cultured in standard medium with 95% O2 and 5% CO2, without additional treatment at 37°C.
3. qRT-PCR
Total RNA extraction from HT22 cell groups utilized the TRIzol kit by Invitrogen (USA), followed by cDNA template generation using the Takara reverse transcriptase kit (RR047A, Japan). Sample preparation employed the Takara SYBR® Premix Ex Taq™ II kit (DRR081, Japan), and RNA quantification utilized the Toyobo SYBR Green real-time PCR Master Mix (Japan) with the Q6 QuantStudioTM 12 K Flex real-time PCR system from Thermo Fischer Scientific (USA). Fluorescence activation was at 95°C for 10 minutes, followed by 40 cycles of denaturation at 94°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 70°C for 30 seconds. All procedures followed MIQE guidelines and were triplicated. Data analysis employed the 2 − ΔΔCt method, with GAPDH as the mRNA standardization reference and U6 for miRNA. Primer sequences are provided in Supplementary Table 1, with all primers sourced from Shanghai GenePharma Co., Ltd. The primer sequences are displayed in Supplementary Table 1.
4. Measurement of MDA, ROS, LDH, GSH, and intracellular ferrous ions (Fe2+) levels.
Following the manufacturer's protocol, MDA, LDH, GSH, and Fe2 + levels were evaluated in HT22 cells and their supernatants. Initially, HT22 cells were seeded into a 96-well plate and centrifuged at 4°C for 10 minutes at 13,000 × g post-treatment with an iron reagent buffer. Next, 50 µL each of supernatant and buffer were mixed and incubated at 25°C for 30 minutes. Then, 200 µL of 2′,7′-dichlorodihydrofluorescein diacetate probe was added, followed by a 30-minute co-incubation at 25°C in the dark. Fluorescence intensity was measured at 485nm excitation and 525nm emission wavelengths using a fluorescence microplate reader. ROS levels were expressed as a percentage, calculated by dividing the fluorescence value of the treated group by that of the control group and multiplying by 100%.
5. Cell counting kit 8 (CCK-8)
Cell viability was evaluated utilizing the CCK-8 assay kit procured from Beyotime in Shanghai, China. Following successful transfection, HT22 cells were harvested and distributed into a 96-well plate at a density of 1×10^5 cells/mL, with each well containing 100 µL of dense medium. Subsequently, the CCK-8 reagent was introduced into each well, and the cells were incubated in a 37°C culture chamber for 4 hours. Cell viability was then determined by measuring the absorbance at 450 nm using a microplate reader.
6. TUNEL assay
To evaluate HT22 cell death, the TUNEL detection kit from Beyotime Biotech was used following the manufacturer's instructions. After OGD/R injury, HT22 cells underwent two PBS washes, followed by the addition of 50µL TUNEL detection solution and a 1-hour incubation at 3°C in darkness. Subsequently, the cells were resuspended in 500µL PBS and examined under a fluorescence microscope, with excitation wavelengths of 460nm (green channel) and 565nm (blue channel).
7. Western blot
HT22 cell proteins are initially extracted using RIPA reagent and quantified with the BCA assay. Following separation on a 15% SDS-PAGE gel, the proteins are transferred to a PVDF membrane. Next, the PVDF membrane is treated with 5% skim milk for 2 hours and then incubated overnight at 4°C with primary antibodies targeting ferroptosis-related proteins such as TFR1, GPX4, and GSH, while GAPDH serves as the loading control. Afterward, the membrane undergoes a 1-hour incubation with secondary antibody complexes (either anti-mouse IgG or anti-rabbit IgG). Protein expression levels are then detected using the ECL chemiluminescence method, with all experiments conducted in triplicate.
8. Dual-luciferase reporter assay
Following the described protocol, we constructed recombinant luciferase reporter plasmids harboring miR-6838-5p binding sequences for circGRB10 and FBXW4, alongside recombinant plasmids containing wild-type (WT) and mutant (mut) 3'-UTR regions. HT22 cells were seeded in 12-well plates, transfected with either miR-6838-5p mimic or miR-NC mimic, and co-cultured with circGRB10 and FBXW4 WT or mut for 48 hours. Subsequently, cellular lysis was conducted, followed by the evaluation of luciferase activity using the RG027 luciferase reporter assay kit, wherein the activity of firefly luciferase was standardized against that of Renilla luciferase.
9. RNA pull-down assay
The RNA pull-down assay utilized the MagCapture™ RNA Pull-Down Assay Kit. Initially, 500 µg of streptavidin-coated magnetic beads were incubated with 200 pmol of biotin-labeled miR-6838-5p mimic in RNA extracted from HT22 cells. After gentle mixing and a 30-minute incubation at room temperature, the streptavidin-coated magnetic beads were left overnight at 4°C to capture the target RNA. Subsequently, the magnetic beads were separated from the protein complex, and the pulled-down RNA complex was collected for validation using qRT-PCR.
10. Cell transfection
HT22 cells were transfected with si-circGRB10 1#, si-circGRB10 2#, miR-6838-5p mimic/inhibitor, FBXW7, and corresponding negative control plasmids (Abiocenter Biotech, USA) using Lipofectamine® 3000 reagent (Invitrogen, USA) according to the manufacturer's protocol.
11. Bioinformatics
From the Gene Expression Omnibus database, we acquired a microarray profile, GSE133768, pertinent to acute ischemic stroke. Rigorous screening ensued to discern all differentially expressed circular RNAs (circRNAs), employing established thresholds of statistical significance (P < 0.05 and |log2 + FC| ≥ 1.0). Furthermore, computational tools, including Starbase and TargetScan, were utilized to forecast potential binding sites between messenger RNA (mRNA) and microRNA (miRNA).
12. RNA immunoprecipitation (RIP) assay
The interaction between circGRB10 and miR-6838-5p was evaluated using a RIP Kit (#17–700, Millipore, USA). Cell lysate was divided, with one part used as input and the other part incubated with antibody for Co-IP. Following washing and centrifugation, RNA was extracted by proteinase K treatment. circGRB10 and miR-6838-5p levels were then quantified via qRT-PCR. In the RIP assay, samples were treated with 8 µg anti-human Argonaute2 (AGO2) antibody (MA5-23515, Invitrogen, USA) and 10 µl recombinant anti-human IgG (EPR442, Abcam, USA) for 30 min, with recombinant anti-human IgG as a control.
13. Statistical analysis
Data analysis employed GraphPad Prism 8.3 software, presenting results as mean ± standard deviation. Statistical significance was determined via Student's t-test for pairwise comparisons and one-way ANOVA, followed by Tukey's post hoc test for multiple comparisons. Findings were deemed significant at p < 0.05. Crucially, experiments were conducted in triplicate to ensure robustness and reproducibility.