Tissues and serum specimen collection This study has been approved by the Ethics Committee of the Nanjing Medical University Affiliated Cancer Hospital and was performed in accordance with the provisions of the Ethics Committee of Nanjing Medical University. We obtained the written informed consent from all the patients. 40 paired Human BCa tissues, ANT were obtained from the Department of Thoracic Surgery, Jiangsu Cancer Hospital between 2010 and 2016 (Nanjing, China).
Cell cultures The BIU-87 cells and 5637 cells were obtained from the Chinese Academy of Sciences Cell Bank and were authenticated by the providers by DNA-fingerprinting analysis or isoenzyme analysis, and were tested negative for mycoplasma contamination. BIU-87 cells and 5637 cells were cultured in RPMI-1640 medium (Keygen Biotech, Nanjing, China) supplemented with 10% fetal bovine serum (Gibco, Grant Island, USA). Cells were maintained in an atmosphere of 5% CO2 in a humidified 37°C incubator. Cells were authenticated by STR analysis at Guangzhou Cellcook Biotech Co., Ltd. (Guangzhou, China) Characterized Cell Line Core Facility within the last three years and routinely tested negative for mycoplasma contamination.
Over-expression or knockdown of genes Human circAPP linear sequence was obtained from esophageal squamous cell carcinoma tissues by PCR and inserted into plasmid vector pcDNA 3.1 (Hanbio, shanghai, China). Human APP cDNA was amplified with PCR primers and subcloned into pcDNA3.1 empty vector (Hanbio). The small interfering RNA (siRNA) of circAPP and mAPP were provided by RiboBio (Guangzhou, China). The transient transfection of the overexpressing plasmids were performed using the Lipofectamine 3000 kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions, and the transient transfection of siRNA were performed using the Lipofectamine iMax kit(Invitrogen) according to manufacturer’s instructions.
RNase R treatment & Quantitative PCR. Total RNA was isolated from cells and tissues using Trizol reagent (Life Technolohies, Scotland, UK) according to the manufacturer’s protocol. And the RNA was extracted from serum with miRNeasy Mini Kit (Qiagen, Hilden,Germany). Nuclear and cytoplasmic RNA was extracted using nuclear and cytoplasmic RNA purification kit (Fisher scientific, Vilnius, Lithuania). For RNase R treatment, 1 µg of total RNA was incubated 30 min at 37°C with or without 3U of RNase R (Geneseed, Guangzhou, China). Reverse transcription was then performed using random hexamers (Takara, Dalian, China) and quantitative PCR (qPCR) was performed using SYBR Green master mix (Applied biosystems, Vilnius, Lithuania). To quantify expression of circRNA transcripts, divergent primers were designed to amplify across the back-splicing junction. Amplification was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA) and Ct thresholds were determined by the software. Expression was quantified using 2-ΔΔCT method using GAPDH (for mRNA/ circRNA) or U6 small nuclear RNA (for nuclear RNA fraction) as reference genes.
Western blotting Briefly, total protein of cells was extracted using RIPA (Thermo Fisher Scientific, Waltham, USA) with a cocktail of proteinase and phosphatase inhibitors (Thermo Fisher Scientific) according to its protocol. Equal amounts of protein lysates were resolved by SDS-PAGE gels and then transferred on a PVDF membrane (Millipore, Massachusetts, USA). After incubation with a primary antibody at 4°C overnight, the membranes were hybridized with a secondary antibody at room temperature for 1 h. Blots were visualized using ECL detection (Thermo Fisher Scientific).
Transwell and Matrigel assay For migration assay, 4 × 104 cells were seeded into the upper transwell assay chambers with 8µm pore filters (Millipore) in serum-free medium. For invasion assay, 4 × 104 cells were seeded into the upper matrigel assay chambers with a matrigel-coated membrane (Corning, Massachusetts, USA) in serum-free medium. The lower chamber contained medium with 10% FBS as chemokine. After incubation for 24 hours for migration and 48h for invasion at 37℃, non-migrating or non-invading cells were gently removed and cells migrated to the bottom of the membrane were fixed with 4% paraformaldehyde, stained with crystal violet solution for 30 min, and visualized under a microscope at × 100 magnification.
Wound-healing assay Transfected cells were cultured in 6-well plates. After the cells reached 90% confluence, a standard 200µl pipette tip was subsequently utilized to scratch linear wounds. In addition, the cell monolayers were cultivated in FBS-free medium. After scratching, the images of the wound closure were captured at 0, and 36h.
RNA pull-down The Biotin-labled RNA probes of circAPP and scramble were synthesized by GenePharma Company (Suzhou, China). RNA pull-down assay was performed using a Biotinylated Protein Interaction Pull-Down Kit (Thermo Fisher Scientific). In brief, 2×107 cells incubated in lysis buffer on ice for 30min. The streptavidin-coated magnetic beads were incubated with biotinylated probes at room temperature for 30 min. The beads-probe complex was added to lysis, and mixed at 4℃ for 2h. The bound miRNA were eluted from the packed beads. The miRNA in the capture complex were identified by qRT-PCR.
RNA-Fluorescence in situ hybridization assay and Fluorescence immunocytochemical staining RNA-Fluorescence in situ hybridization (FISH) assays were performed using a RNA-FISH kit (GenePharma, China) according to the manufacturer’s instructions. Cy3-labeled antisense probe was synthesized by GenePharma company (Suzhou, China) against the junction site of circAPP. In briefly, 5637 cells were fixed with 4% paraformaldehyde. After pre-hybridization with 1× PBS/0.5% Triton X-100, cells were blocked and hybridized in hybridization buffer with Cy3-labeled probe at 37°C overnight. Cells were stained with DAPI (300 nmol/L).
Statistics All statistical analyses were performed with SPSS 25.0 software. Qualitative variables were analyzed by chi-square test or fisher’s exact test. For continuous variables, if which obey the normal distribution, student’s t test is used to compare the differences. Otherwise, variables were compared using nonparametric test for which with an abnormal distribution. Differences between groups were compared using analysis of variance (ANOVA) when applicable or a nonparametric test. Correlation analysis was performed using the Pearson correlation coefficient method. Unless otherwise specified, the results are presented as the means ± standard deviation (SD). All statistical tests were 2 sided, and P < 0.05 was considered statistically significant.
The circRNA microarray is available in the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) under accession numbers GSE147985). The source data of other figures are provided as a Source Data file. All other data are available from the authors upon reasonable requests.