Microarray
The miRNA microarray GSE135918 (Jiang N et al. 2020) was obtained from the GEO database. There was 5 lung cancer tissues and 5 non-tumor lung tissues in the microarray. The differentially expressed miRNAs were analyzed using GEO2R.
Cell lines and cell transfection
Four human lung cancer cell lines (HCC827, A-549, Calu-3, and H1975) and one normal lung epithelial cell line and BEAS-2B were bought from iCell Bioscience Inc (Shanghai). The cells were seeded in the DMEM medium contain 10% FBS (Gibco, Waltham, MA) and 1% Pen/Strep solution under 5% CO2 at 37°C.
For cell transfection, small interfering RNA (siRNA) target hsa_circ_0078767 and TP53, as well as the hsa_circ_0078767 overexpression vector was produced by GenePharma (Shanghai, China). The mimics/inhibitor for miR-330-3p and their corresponding negative controls were from Genechem (Shanghai, China). Transfection action was performed using Liposome 2000 (Invitrogen, USA).
qRT-PCR
Total RNA was extracted with TRIZOL(Invitrogen). The PrimeScript RT Reagent (TaKaRa, Japan) is employed to reverse transcribed RNA into cDNA. A 7500 RT-PCR System (Applied Biosystems, Carlsbad, CA, USA) with a SYBR Green Master Mix (Roche, Shanghai, China) were employed for subsequent quantitative RT-PCR. RNA expression fold changes were calculated with 2−ΔΔCt method. β-actin and U6 were applied as the template normalization. The primer sequences were listed in Table S1.
RNase R and Actinomycin D treatment
A-549 and H1975 cells were incubated with RNase R and actinomycin D to verify the circRNA characteristics. For RNase R treatment, 3 µg RNA was mixed with 20 U/µL RNase R and incubated at 37℃ for 15 min for reaction. For actinomycin D treatment, the cells were incubated with actinomycin D for 0 h, 6 h, 12 h, 18 h and 24 h. Then, hsa_circ_0078767 and FAM120B expression were detected by qRT-PCR.
RNA fluorescence in situ hybridization (FISH)
Specific probe against hsa_circ_0078767 was applied for FISH assay. The procedures were performed according to the manufacturer’s instructions (GenePharma, Shanghai, China). In short, after paraformaldehyde (PFA) fixation, PBS washing, ethanol dehydration, cells were kept overnight at 37°C for hybridization. Then, after washing for 3 times, cells were incubated in blocking buffer for 1 h. After incubation with an HRP-conjugated anti-biotin antibody at 4°C overnight, images were obtained with a fluorescence microscope.
CCK-8 assay
In brief, cells were seeded in 96-well plates (1 × 103 cells/well). Then, 10 µL of CCK-8 solution (Dojindo, Japan) was added to each well. After incubation, absorbance was tested at 450 nm with a microplate reader (Bio-Rad, CA, USA).
Colony formation assay
A density of 5 × 103 cells/well A-549 and H1975 cells were seeded into six-well plates. 14 days later, the colonies were fixed with 4% PFA, and counted after 0.1% crystal violet staining.
5-Ethynyl-2′-deoxyuridine (EdU) staining
DNA synthesis and cell proliferation were detected by EdU assay kit (RiboBio, China). A-549 and H1975 cells were cultured at 50 mM EdU for 2 h, then immobilized with 4% PFA, stained with Apollo Dye, and finally stained with DAPI. Then, the EdU positive cells were photographed under a microscope (Nikon, Japan).
Flow cytometer apoptosis analysis
After transfection, A-549 and H1975 cells were harvested, washed, and suspended in 600 µL flow cytometry binding buffer. Next, cells were incubated with Annexin V-FITC (5 µL) and PI (5 µL). After incubation, cell apoptosis was examined by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
Measurement of iron and ROS
A-549 and H1975 cells were first treated with 5 mmol/L erastin or 1 mmol/L ferrostatin. Cell vitality was analyzed by CCK-8 as described above. Cell iron and Fe2+ levels was measured by Iron Assay Kit. The content of ROS was examined by flow cytometry.
Western blot
RIPA buffer (CST, USA) was employed to lyse cells and harvest protein. The same amount of protein was electrophoresed on SDS-PAGE (12% polyacrylamide gel) and then transferred to PVDF membrane (Millipore, USA). After being blocked with 5% milk, the membranes were incubated with Cleaved Caspase 3 (AB2302, 1/500, Abcam), cleaved PARP (AB32064, 1/2000, Abcam), p53 (AB32389, 1/10000, Abcam), SLC7A11 (AB175186, 1/3000, Abcam), GPX4 (AB125066, 1/2000, Abcam) and β-actin (AB8227, 1/2000, Abcam) and corresponding second antibody. The results were then observed with an enhanced chemiluminescence detection system.
RNA pull-down assay
A-549 and H1975 cells were harvested for lysed. To generate probe-coated beads, hsa_circ_0078767 probe was incubated with C-1 magnetic beads (Life Technologies) at room temperature for 2 h. Cell lysate containing hsa_circ_0078767 probe or oligo probe was incubated at 4°C overnight. Then, the bound RNA was washed and extracted with a RNeasy Mini Kit (QIAGEN) for qRT-PCR detection.
RNA binding protein Immunoprecipitation (RIP) assay
The Magna RIP kit (Millipore, Billerica, MA, USA) was applied for RIP assay. In simple terms, cells were collected and the nuclei cleaved to fragment chromatin, followed by immunoprecipitation with RIP buffer containing magnetic beads coated with anti-Ago2 antibody (Millipore, Billerica, MA, USA), or negative control IgG. After incubation, cells were collected and the nuclei cleaved to fragment chromatin, followed by qRT-PCR detection.
Dual-luciferase reporter assay
Dual-luciferase Reporter Assay System (Promega, USA) were applied for the luciferase reporter gene assays. In brief, the A-549 and H1975 cells were treated with the miR-330-3p mimic, or control mimic, pmirGLO-hsa_circ_0078767, pmirGLO-hsa_circ_0078767 mutant, pmirGLO-TP53, and pmirGLO-TP53 mutant using Lipofectamine 2000 (Invitrogen, USA), The luciferase activities was measured and normalized to the activity of Rluc.
In vivo xenograft models
Six-week-old male nude mice were used in the current study. A-549-vector and A549-oe-circ cells (1 × 106 cells) were subcutaneously injected into the flanks of the nude mice, Tumor length and width were measured every 7 days and tumor volume was calculated: volume = length × (width/2)2. 21 days after injection, the mice were sacrificed and the tumor were taken out and weighted. All experiments were approved by the Ethics Committee of Kunming Medical University of Yunnan Province.
Statistical analysis
Data are presented as the mean ± SD. All statistical analyses were conducted using GraphPad Prism 9.3.1. Differences between groups were compared by student’s t-test or one-way ANOVA test. P < 0.05 was considered to be statistically significant.