Patient samples
Samples of 173 lung cancer patients were collected from Sun Yat-sen University Cancer Center (SYSUCC), Guangzhou, China between January 2013 and December 2014. Among them, 161 paired lung cancer and adjacent normal tissues were used for the verification of expression level of circSCAP. All patients were free from neoadjuvant therapy before surgery. Clinic pathological features included age, gender, histological type, T stage, N stage, TNM stage, vessel invasion and adjuvant treatment were recorded. The median follow-up time was 32.5 months (range: 1–80 months). Our study was approved by the Ethics Committee of SYSUCC, the reference number is GZR 2018–120. Informed consent was obtained from each patient.
Cell culture
Human NSCLC cell lines H1650 (wild-type p53), A549 (wild-type p53), H1299 (p53 null), PC9 (inactivating p53 mutation) and HCC827 (inactivating p53 mutation) and the normal human lung epithelial cell line 16HBE were purchased from the American Type Culture Collection (ATCC, Manassas VA, USA), authenticated by short tandem repeat (STR) profiling, and resuscitated within 6 months. The cell lines were cultured in RPMI 1640 (Gibco), containing 10% FBS (Gibco) and 1% penicillin–streptomycin at 37℃ in a humidified air with 5% CO2. Antibiotics were removed during validation and characterization experiments.
Cell transfection
Lentivirus-circSCAP and lentivirus-vector were purchased from GenePharma (Suzhou, China). Lentiviruses were ultracentrifuged, concentrated, and validated. A specific small interfering RNA for hsa_circ_0065214 was designed to target the covalent closed junction. All si RNAs, si NC (negative control), microRNA (miRNA) mimics and inhibitors were also purchased from GenePharma (Suzhou, China) (Table S1). PcDNA3.1( +)-SF3A3 (Umine Biotechnology Co., LTD., Guangzhou, China), pmirGLO- hsa_circ_0065214 (GeneCopoeia, USA), and pp53-TA-luc (Beyotime Biotechnology, Shanghai, China) and pySEAP (Kelei biological Technology Co., LTD., China) were transformed into DH5α competent cells, coated plates, and selected monoclonal colony to multiply. All of them were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).
RNA extraction, quantitative and real-time PCR (qRT-PCR) detection
The total RNA was extracted from collected cells using TRIzol. RNA concentration was measured using NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Then, 2 µg of total RNA was reverse transcribed in a final volume of 20 µL with the Prime Script RT Master Mix (ESscience, Shanghai, China). qRT-PCR was then performed with SYBR Premix Ex Taq TM (Yeasen, Shanghai, China) on Light Cycler 480 (Roche, Switzerland). The expression levels of mRNA were determined by the comparative cycle threshold (CT) (2-∆∆Ct) methods (mRNA and miRNA were normalized by GAPDH and U6, respectively). All the PCR primers were listed in Table S2.
Cell proliferation assay
CCK8 (Yeasen, Shanghai, China) assay was performed 48 h after transfection. The A549 and H1650 cells were cultivated on a 96-well plate (1000 cells per well) for 1–6 days. SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices LLC, Sunnyvale, CA, USA) was then employed after an hour of incubation with CCK8 solution at the absorbance of 450 nm.
Cells were made into single-cell suspensions and then incubated in six-well plates at a density of 1000 cells per well for cell colony formation assay. After 2 weeks, cells were fixed in methanol and stained with 0.1% crystal violet (Weijia Biology Science and Technology Co., Guangzhou, China) for 20 min. After washed by PBS, images were captured under a microscope.
EdU proliferation assay (RiboBio, China) was conducted to detect the proliferation of the transfected cell. The cells were fixed by 4% paraformaldehyde for 20 min after being treated with 50 µM EdU for 2 h. Following Apollo staining and Hoechst33342 staining, the fluorescent microscope was applied to observe the EdU positive cells.
Wound healing assay
This essay aimed to assess the migration abilities of cells after transfection. The cells were seeded in a six-well plate. After 24 h transfection, when the cells were dense in the field of microscopic view, three standardized wound scratches per well were generated with a sterile 20 µL pipette tip, and the 10% FBS medium was replaced with serum-free RPMI 1640. A phase-contrast microscope was employed to photograph the width of the scratch at different time frames (0,12 h and 24 h).
Transwell assay
Migration assays were executed using a Transwell chamber with 8 µm pores (Costar, Corning, New York, NY, USA). Cells (1.0 × 105) in 200 µL of serum-free RPMI 1640 medium were seeded in the upper chamber placed in the 24-well culture plate, and the lower chamber contained 700 μL medium with 10% FBS as a chemoattractant. The chambers were maintained at 37 °C and 5% CO2 for 16 h. Those invaded cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet (Weijia Biology Science and Technology Co., Guangzhou, China) for 30 min. A phase-contrast microscope was applied to count the number of stained cells (5 different views per well).
Flow cytometry analysis
For the cell apoptosis, the cells were suspended by trypsin without EDTA and washed by phosphate-buffered saline (PBS) thrice. 5 µl of FITC Annexin V and 5 µl of propidium iodide (biogems, CA, USA) were immediately added to the transfected cells suspending in 500 µl of binding buffer for 15 min in dark and then examined with flow cytometry. In the cell cycle analysis, the cells seeded in 6-well-plates for 60–70% confluence was synchronized at the G0 boundary by serum-free medium for 24 h before transfection. After staying in the 70% ethanol at 4℃ overnight, the cells were stained in dark for 15 min 500 µl propidium oxide staining solution (Yishan, Technology Co., Shanghai, China), and then detected by a flow cytometer (NCEA).
Western blot
The total proteins were collected from cells using RIPA Lysis Buffer containing 1% phenylmethylsulphonyl fluoride (PMSF) (Beyotime, Shanghai, China), and the protein concentration was measured with a BCA protein assay Kit (Beyotime, Shanghai, China). Protein were denatured at 100 °C for 8 min with DualColor Protein Loading Buffer (Life, USA). The denatured proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated for 20 min in quick blocking solution (Beyotime, Shanghai, China) at room temperature followed by incubation with primary monoclonal antibodies overnight at 4 °C with soft shaking. The primary antibodies against human p53, SF3B2, SF3B3, RBM17, Lamin-B1 and GAPDH were purchased from Proteintech Group (IL, USA). SCAP was purchased from Cell Signaling Technology (Danvers, MA 01,923, USA). CDK2/4/6, Cyclin D1, SF3A3, and p53 S46 were purchased from Abcam (Cambridge, MA, USA). And enhanced chemiluminescence reagent kit (Affinity Biosciences, Jiangsu, China) was utilized for exposure after the blot incubated with secondary antibody (Proteintech) for 1 h.
Tumor xenograft experiments
Nude mice (BALB/c, SPF grade, male, 4–5 weeks old) were obtained from Beijing Vital River Laboratory Animal Center (Beijing, China), and were maintained under specific pathogen-free conditions. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use of SYSUCC. Mice were randomly divided into different study groups, and subcutaneously implanted with 2 × 107 H1650 cells or 4 × 107 A549 cells respectively. Tumor sizes were measured every 3 days using calipers, and the tumor volumes (VT) were calculated according to the formula: VT = (length) × (width)2/2. Finally, the mice were euthanized and the subcutaneous tumors were resected for subsequent use.
Immunohistochemistry
Formalin‐fixed and paraffin‐embedded tissue sections were incubated with Ki67 primary antibody (dilution 1:1000; Proteintech), PCNA antibody (dilution 1:300; Proteintech), and SF3A3 (dilution 1:200; Abcam) overnight at 4 °C. Then, the sections were incubated with HRP-conjugated anti-rabbit IgG secondary antibody (dilution 1:300; Yeasen) for 2 h at room temperature. The slides were evaluated by two independent observers.
Luciferase reporter assay
The sequence of circSCAP was cloned downstream of p-GLO Dual-Luciferase vector (Jidan, Guangzhou, China). Mutations were performed in the binding sites. The cells were cultured in a 24-well plate until showing 60–70% confluence, and then the constructed report vectors containing wild-type fragment or mutant type fragment together with renilla vector and miRNA mimics or miR-NC were co-transfected into 293-T cells using LipofectamineTM 3000 reagent. The cells were collected after 48 h for luciferase detection with the dual-luciferase reporter gene assay system (Promega, Madison, WI, USA). The firefly luciferase activity was normalized based on Renilla luciferase activity.
p53 signaling activity assays
Si SF3A3 transfection two days later, pp53-TA-GLuc-Dura, pGLuc-Dura-TA and pySEAP reporter plasmids were transfected with addition of 10 μM pifithrin-α, and luciferase was measured 24 h later.
RNA pull-down
The biotin-coupled RNA complex was pulled down by incubating the cell lysates with streptavidin-coated magnetic beads following the manufacturer’s instructions (Axl-bio, Guangzhou, China). The bound proteins were eluted from the packed beads and analyzed by SDS-PAGE. CircSCAP probe and control probe are shown in the Table S3. The probe solution was denatured at 90 °C for 2 min and then incubated with pre-cooled RNA structure buffer to form RNA secondary structures. Afterwards, streptavidin magnetic beads were incubated with the mixture at 250℃ for 30 min. Cells were lysed at 4℃ and centrifuged. Subsequently, the supernatant fractions were collected and mixed with the probe-bead mixture. After incubation and elution at 37℃ for 2 h, the proteins in the capture complex were identified by western blotting or mass spectrometry analysis.
RNA immunoprecipitation (RIP) assay
RIP assay was performed using RIP-Kit (BersinBio, Guangzhou, China) according to the manufacturer’s instructions. The obtained RNA was extracted by SF3A3, human anti-Ago2 antibodies (Abcam, ab32381, Shanghai, China), or negative control IgG (Beyotime, Shanghai, China), respectively.
RNA-FISH
Biotin-labeled antisense or sense probe for circSCAP junction and U6 were synthesized (Exon Bio, Guangzhou, China) (Table S3). Cells were incubated with 40 nM FISH probe in circRNA hybridization buffer at 37 °C for 36 h. After being washed, cells were incubated with anti-digoxin HRP conjugate at 37 °C for 1 h. Then cells were incubated with TSA (Exon Bio, Guangzhou, China) in dark for 15 min, and sealed with DAPI. The images were acquired using a fluorescence microscopy (OLYMPUS FV1000 confocal microscopy, Japan).
Nuclear and cytoplasmic extraction
Cytoplasmic and nuclear fractions were isolated according to the manufacturer’s manual, using the reagents supplied in Invent Kit (Invent Biotechnologies, Beijing, China). Briefly, the cells were lysed in Cell Fraction Buffer on ice for 10 min. After centrifugation at 14000 rpm for 5 min at 4 °C, the supernatant was collected as cytoplasmic fraction, and then the nuclei were collected by washing the pellet with Cell Fraction Buffer.
Immunoprecipitation (IP)
Transfected cells were lysed with Pierce™ IP Lysis Buffer (Invitrogen, CA, USA). Protein concentrations were measured with a BCA protein assay kit (Beyotime, China) and the lysates were incubated with primary antibody for Ubiquitin HA-tag (Abcam) or SF3A3 (Abcam) with protein A/G magnetic beads (MedChemExpress, USA) at 4 °C overnight. The beads were washed using IP lysis buffer for 6 times before immunobloting by the indicated antibodies.
Statistical analysis
SPSS 20.0 (IBM, Armonk, NY, USA) and Graphpad Prism 7 (GraphPad Software Inc., CA, USA) was used for data analysis. Image J software was used to analyze cell migration, and protein expression. The data are shown as the means ± SD from at least three independent experiments. Comparisons between groups for statistical significance were performed with the two-tailed Student’s t test or two-way ANOVA. Paired sample t test and Mann–Whitney U test were used to compare the relative expression of circSCAP in lung tumor and peritumor tissues. Chi-square test or Fisher’s exact test was applied to study the relationship between circSCAP expression level and clinicopathological variables of lung cancer patients. The survival curves were plotted using the Kaplan–Meier method. Cox regression analyses were used to study the correlations between variables and survival. P < 0.05 was considered statistically significant.