Cell culture and transfection
NSCLC cell lines (A549, PC9, H838, H1299, 95D), human normal lung epithelial cells (BEAS-2B), and 293A cells were purchased from Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM medium (Gibco, CA, USA) containing 10% fetal bovine serum (FBS) in a humidified incubator containing 5% CO2 at 37 °C. For hypoxia studies, the cells were cultured at 37 °C under 1% O2 and 5% CO2 in a humidified hypoxic chamber (Forma Scientific, Marietta, OH, USA).
Small interfering RNAs (siRNAs) targeting circ-0001875 and SP1, miRNAs mimics, inhibitors and corresponding negative controls were purchased from GenePharma (Shanghai, China). The full-length cDNA sequence of circ-0001875 and SP1 were used to construct overexpression plasmids by GenePharma. All siRNAs, miRNA mimics and inhibitors were transiently transfected into cells with Lipofectamine RNAiMAX (Invitrogen), and the overexpression plasmids were transfected with Lipofectamine 3000 (Invitrogen). The circ-0001875 overexpression (pLCDH-circ-0001875) and knockdown vector (sh-circ-0001875) and the control vector (GenePharma) were transfected into A549 and H1299 cells. Cells were selected with puromycin (Sigma-Aldrich, USA) for 2–3 weeks to establish stable cell lines. Sequences of all constructs used in this study are listed in Table S1.
Tissue samples
We acquired 72 pairs NSCLC and matched tumor-adjacent tissues from patients who underwent surgery without preoperative chemotherapy or radiotherapy at Affiliated Hospital of Guangdong Medical University between 2017 and 2020. All tissues were stored in liquid nitrogen. This research was approved by the Ethics Committee of Affiliated Hospital of Guangdong Medical University. All patients signed informed consent forms before research was conducted.
Animal models
Four-week-old female nude mice (BALB/c) were purchased from the medical experimental Animal Center of Guangdong province (Guangdong, China).
For the subcutaneous tumor model, the armpits of mice were subcutaneously injected with transfected cells (6 × 106 cells/mice, n = 9/group for overexpression groups, n = 6/group for knockdown groups). In one experiment, mice were randomized into two groups: control shRNA (sh-NC) and circ-0001875 shRNA (sh-circ-0001875). In a second experiment, mice were randomized into two groups: control vector (pLCDH-vector) and circ-0001875 overexpression vector (pLCDH-circ-0001875). Tumor growth was monitored and measured with a vernier caliper every week. Tumor volume was calculated with the formula: V = (width2 × length)/2. At 4 weeks after tumor formation, mice were euthanized and subcutaneous tumors were harvested for HE and IHC staining.
For metastasis studies, transfected cells were injected into the tail vein of mice (10 × 106 cells/mice, n = 9/group for overexpression groups, n = 6/group for knockdown groups). After 6 weeks, lungs and livers were dissected, and all samples were weighed.
Animal experiments were approved by the animal ethics committee of Guangdong Medical University and were fed in the SPF grade of the Laboratory Animal Center of Guangdong Medical University.
RNA-seq
Total RNA was isolated from three pairs of NSCLC tissues and adjacent normal tissues by TRIzol Reagent (Invitrogen, CA, USA). The RNA integrity was assessed by Agilent 2100, samples with an RNA integrity number > 7.0 were used in experiments. Ribosomal RNA was removed from samples using a RiboMinus Eukaryote kit (Qiagen, Valencia, CA, USA). The RNA-seq library was deep sequenced using an Illumina HiSeq 2000 instrument (Illumina, San Diego, CA, USA) to harvest paired-end reads, and Edger software (v3.16.5) was used to identify the differentially expressed circRNAs.
RNA extraction and quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA was extracted from tissues and cells by Trizol reagent (Invitrogen) in accordance with the manufacturer’s instructions. cDNA was synthesized using Evo M-MLV RT Premix (AG, Hunan, China) and RT-qPCR was performed on an ABI7500 (Applied Biosystems, Foster City, CA, USA) or LightCycler 480 (Roche Applied Biosystems). U6 and β-actin mRNA were used as internal loading controls for miRNA and mRNA, respectively. Primers (Sango Biotech, Shanghai, China) used in the study are listed in Table S2. Each sample was replicated at least three times and data were analyzed by the 2-ΔΔCT statistical method.
RNase R treatment and actinomycin D (ActD) treatment
For RNase R treatment, total RNA extracted from cells (2 μg) was incubated at 37 °C for 15 min with 3U/μg RNase R (Epicentre Technologies Corporation, Madison, WI, USA). For ActD treatment, cells were cultured with 2 μg/mL ActD (Beyotime, Shanghai, China) for specific times.
Cell Counting Kit-8 (CCK8) assay and cloning formation assay
For CCK-8 assay (Beyotime), transfected cells were inoculated into 96-well plates at 2000 cells in 100 μl medium per well. At 0, 24, 48, 72 and 96 h after inoculation, 10 μl of CCK-8 reagent was added and cells were incubated at 37 °C for 1 h. Cell viability was detected at absorbance at 450 nm.
For cloning formation assay, transfected cells (500 cells/well) were inoculated into 6-well plates and cultured at 37 °C for 2 weeks. Cells were fixed in methanol for 30 min and then cells were stained with 0.1% crystal violet. The cells were imaged and counted.
Wound healing assay
Cells were inoculated into 6-well plates and transfected. Scratch wounds were generated in the cell monolayer by 1-ml pipette tip after cell density reached approximately 90%. Floating cells were removed by washing with 1 × PBS, and cells were incubated in serum-free medium. Wound healing was photographed at 0, 24 and 48 h using inversion microscopy. Cell migration was analyzed by ImageJ.
Migration and invasion assays
Approximately 3 × 105 transfected cells in 200 μl serum-free medium were inoculated into the upper chamber of a Transwell chamber (BD Biosciences, Franklin Lakes, NJ, USA) with or without Matrigel (BD Biosciences) for invasion and migration assays, respectively. The lower chambers were filled with 750 μL medium containing 20% FBS. After incubation of culture plates for 24 h, cells were fixed with methanol and stained with 0.5% crystal violet (Beyotime). Migrated and invaded cells were imaged under a microscope.
Western blot
Transfected cells were lysed in RIPA (Beyotime) containing PMSF and protein concentrations were quantified with BCA reagent (Beyotime), Equal amounts of protein were electrophoresed on 10% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking the membranes with 5% defatted milk at room temperature for 1 h, the membranes were incubated with primary antibodies at 4 °C on a shaker overnight. The membranes were then incubated with indicated HRP-labeled secondary antibodies for 1 h. Protein signals were detected by BeyoECL star (Beyotime). All antibodies used in this study are listed in Table S3.
Fluorescence in situ hybridization (FISH) and immunofluorescence staining
Cells were fixed with 4% paraformaldehyde and permeabilized in PBS with 0.5% Triton X-100 for 30 min in a confocal dish. For FISH, cells were incubated with FITC-labeled circ-0001875 probe (GenePharma) at 37 °C overnight. To detect filopodia formation, cells were incubated with Phalloidin (YEASEN, Shanghai, China) for 90 min at 37 °C. Anti-fade mounting medium was added with DAPI staining, and signals were imaged by Olympus Laser confocal microscopy (Olympus Corporation, Tokyo, Japan).
RNA-binding protein immunoprecipitation (RIP) assay
RIP assays were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). Approximately 2 × 107 cells were lysed in RIP lysis buffer containing protease and RNase inhibitors. Cell lysates were then incubated with IgG or anti-SP1 antibody conjugated magnetic beads (Millipore) with rotation at 4 °C overnight. The next day, the RNA/bead complexes were washed with RIP wash buffer and resuspended in Proteinase K buffer to purify immunoprecipitated RNA. RNA was reverse transcribed to cDNA and analyzed by RT-qPCR.
Co-immunoprecipitation (CoIP) assay
Transfected cells were lysed with RIPA buffer (Beyotime) and incubated with normal IgG, anti-SP1, or anti-HIF1α antibodies on a rotator at 4 °C overnight. Antibody-protein complexes were captured by incubation with Dynabeads-protein G beads (Beyotime) for 2 h. The complexes were then analyzed by western blot analysis.
Dual luciferase reporter assay
Luciferase reporter plasmids (wild-type and mutant-type) of circ-0001875 and SP1 were synthesized by GenePharma. Cells were inoculated into 24-well plates. When the cell density reached approximately 60%, luciferase plasmids, the Renilla control plasmid and miRNA mimics were transfected. Firefly and Renilla luciferase levels were detected using the Dual Luciferase Assay System (Promega).
Immunohistochemistry (IHC)
Tumor tissues from nude mice were cut into 4 μm slices for slide preparation, the slides were deparaffinized with xylene and rehydrated with ethanol. After incubation with 3% hydrogen peroxide to block endogenous peroxidase activity, the sections were soaked in sodium citrate buffer to recover antigen and 5% BSA was added to block nonspecific binding sites. The sections then incubated with anti-Ki67 and anti-SP1 primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies. Images were photographed by a microscope (Olympus Corporation).
Bioinformatics analysis
Circ-0001875 sequence data were obtained from circBase. The target miRNAs of circ-0001875 were predicted by circular RNA interactome (https://circinteractome.nia.nih.gov). Target genes were predicted by TargetScan (https://www.targetscan.org/), miRbase (https://www.mirbase.org/), and miRDB (https://www.mirdb.org/).
Statistical analysis
GraphPad Prism 8.0 and SPSS 23.0 software were used for statistical analysis. Differences between data of two groups were analyzed by Student’s t-test, while differences between multiple groups were calculated by ANOVA. Survival curves were determined using the Kaplan–Meier method. Correlations between circ-0001875, miR-31-5p and SP1 were analyzed by Pearson’s correlation test. Differences were considered statistically significant at P-value < 0.05.