Tissue samples
In total, we collected 90 fresh NSCLC tissues with paired adjacent non-cancerous lung tissues after obtaining informed patient consent at Renji Hospital affiliated with Shanghai Jiaotong University, China. We evaluated pathological and histological diagnostics of NSCLC based upon the Revised International System for Staging Lung Cancer. Patients received no radiotherapy or chemotherapy prior to tissue sampling. We snap-froze samples in liquid nitrogen and stored them at -80°C before RNA extraction. The Ethics Committee of Shanghai University of Medicine and Health Sciences approved the study.
Strand-specific RNA-Seq library construction and high-throughput RNA-Seq
We extracted total RNA from three paired NSCLC tissues as well as adjacent non-cancerous lung tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). We subjected 3 μg of total RNA from each sample to the VAHTS Total RNA-Seq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to remove ribosomal RNA and different classes of RNA such as non-coding RNA and mRNA. We treated purified RNA with RNase R (Epicentre Technologies, Madison, WI, USA, 40 U, 37°C, 3 h), followed by TRIzol purification. We prepared RNA-Seq libraries using the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) and subjected them to deep sequencing with the Illumina HiSeq 4000 at Aksomics, Inc. (Shanghai, China, accession code: H1712024).
For miRNA and mRNA analysis, A549 cells transfected with siRNA against circ-EPB41 or a negative control (NC) vector were used for high-throughput RNA-Seq as previously described (accession code: H1712024).
Cell lines and culture
We obtained the human normal lung epithelial cell line BEAS-2B and the NSCLC cell lines PC9, A549, H1975 and H1650 from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultivated them in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, Carlsbad, CA, USA) supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum (FBS, Invitrogen) at 37°C in a humidified atmosphere with 5% CO2.
Fluorescence In Situ Hybridization (FISH)
We obtained specific probes for circ-EPB41 (Dig-5′-CATACTGCTCTTGCTCCATCGAGGCTCC-3′-Dig) and miR-486-3p (Dig-5′-CGGGGCAGCUCAGUACAGGAUA-3′-Dig) from Geneseed Biotech (Guangzhou, China). Cells were immunostained with Cy3-conjugated anti-digoxin and FITC-conjugated anti-biotin antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA). We counterstained nuclei with 4,6-diamidino-2-phenylindole (DAPI). Staining was visualized with a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Bioinformatics analyses
We imputed circRNA/miRNA target genes through the Interactome (https://circinteractome.nia.nih.gov/), miRanda (http://www.microrna.org/microrna/home.domiRanda) and circBank (http://www.circbank.cn/) websites. We calculated interactive correlations between miR-486-3p and eIF5A via the Targetscan website.
Cell cycle detection
We dissociated cells in the logarithmic growth phase with 0.25% trypsin (Invitrogen, Carlsbad, CA, USA), resuspended them in PBS and fixed them in 70% ice-cold ethyl alcohol overnight at 4°C. Afterwards, we centrifuged cells at 1200 g for 5 min, resuspended them in 50 μL RNase A (Invitrogen, Carlsbad, CA, USA) and incubated them at 37°C for 30 min. Then, we added 400 μL propidium iodide to the suspension for 30 min, followed by flow cytometric detection (BD Biosciences, San Jose, CA, USA).
Plasmid construction and stable transfection
We synthesized human eIF5A and circ-EPB41 cDNA and then cloned it into the luciferase-labelled pcDNA3.1 vector (Invitrogen). We synthesized wild-type (WT) and mutant (MUT) circ-EPB41 and eIF5A cDNAs and cloned them into pZW1 vectors (Shanghai Institutes for Biological Sciences, Shanghai, China). We also transfected H1650 and A549 cells with the aforementioned plasmids.
Total RNA isolation and the quantitative reverse transcription-polymerase chain reaction (RT-qPCR)
We isolated total RNA from tumor tissues and cells with TRIzol reagent following standard procedures. We examined the concentration and purity of RNA samples spectrophotometrically by capturing the absorbance at 230 nm, 260 nm and 280 nm with a NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA). We deemed OD260/OD280 ratios between 1.8 and 2.1 as acceptable, and also deemed OD260/OD230 ratios >1.8 as acceptable.
We reverse transcribed total RNA before RT-qPCR detection. We obtained primers specific for circ-EPB41, eIF5A and miR-486-3p from GenePharma (Shanghai, China). We performed RT-qPCR using an AB7300 thermo recycler (Applied Biosystems, Carlsbad, CA, USA) with the primers listed below and TaqMan Universal PCR Master Mix. We utilized GAPDH as a reference gene for circRNAs and mRNAs and U6 RNA as an internal control for miRNA expression level. We quantified gene expression via the 2−ΔΔCt method. The primers utilized to assay circ-EPB41 expression were forward, 5′-AGGATCCAAATTTCGATACAGTGGC-3′ and reverse, 5′-ATTTCTTAGCTGCTCGGTAACTGGG-3′. The primers for miR-486-3p were forward, 5′-GCGGGGCAGCTCAGTA-3′ and reverse, 5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACATCCTG-3′. The eIF5A primers were forward, 5′-GAGATGCAGGGGCCTCAGCCACC-3′ and reverse, 5′-GTGATAGGTACCCATCCTGGATG-3′. U6 primers were forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The GAPDH primers were forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-GGATCTCGCTCCTGGAAGATG-3′.
RNA interference and overexpression
The miR-486-3p inhibitors and siRNA against circ-EPB41 (sicirc-EPB41) were purchased from GenePharma. Transfections were performed following the supplier's protocol. In brief, we transferred cells to 6-well culture plates and transfected them using Lipofectamine 2000 (Invitrogen). To achieve eIF5A overexpression, we transfected the pCDNA3.0 vector described above. For xenograft experiments, a lentiviral-mediated circ-EPB41-silencing vector (sh-circ-EPB41) was transfected into A549 cells.
Tumor sphere formation assay
We harvested A549 and H1650 cells and resuspended them as single cells in serum-free medium. After precise cell counting, we added 200 cells/well in 200 μL of serum-free medium to a 96-well plate, 10 wells per group. We changed medium every 2 days. We took images of five randomly selected regions in each group of wells with a camera-equipped microplate reader (Leica, Wetzlar, Germany). We calculated sphere percentage by the number of spheres/200.
Cell proliferation assay
We employed Cell Counting Kit-8 assays (CCK-8, Gibco, Logan, Utah, USA) to quantify cellular proliferation. We seeded transfected cells into 96-well plates at a density of 5,000 cells/well in triplicate. We detected cell viability with the CCK-8 system at 0, 24, 48 and 72 h after seeding, following standard procedure.
For colony formation assays, we seeded transfected cells into 6-well plates at a density of 2,000 cells/well and maintained them in DMEM containing 10% FBS for 10 days. We imaged colonies and counted them after fixing and staining them.
Transwell assay
For invasion assays, we placed Transwell assay inserts (Millipore, Billerica, MA, USA) into 24-well plates. The membrane in the upper Transwell chamber was coated with Matrigel (BD Biosciences). We placed 500 μL of DMEM containing 10% FBS into the bottom chamber and seeded 10,000 cells in 200 μL serum-free DMEM in the upper chamber. After 1 to 2 days, we utilized methanol to fix cells on the membrane and stained them with Crystal Violet. Finally, we observed the cells via a microscope (Leica).
Western blot assay
We extracted protein from tissues or cells with RIPA lysis buffer and performed western blot assays as previously described 19. We obtained primary antibodies against SOX2, OCT-4, Nanog, CD133 and GAPDH from Cell Signaling Technology (Beverly, MA, USA) and stained protein blots following standard procedures. We visualized immunoreactivity with a chemiluminescence detection kit (Western Blotting Substrate, Donghuan Biotech, China).
Immunofluorescence
We fixed cells or tumor tissues in 4% paraformaldehyde for 20 min at room temperature (RT). We then incubated the fixed cells or tissues with 0.5% Triton-X-100 in PBS for five min at RT and blocked them with 5% bovine serum albumin in PBS for 1 h at RT. The cells were incubated with primary antibodies against SOX2 (1:200, Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with fluorochrome-conjugated secondary antibody (1:200, Abcam) and Nestin primary antibody conjugated to FITC (1:200, Millipore) at RT for 1 h. We stained nuclei in DAPI for 15 min at RT and visualized staining via fluorescence microscope.
The 5-Ethynyl-2′-deoxyuridine (EdU) assay
We purchased an EdU assay kit (RiboBio, China) to measure cell proliferation and DNA synthesis. We seeded 10,000 A549 or H1650 cells in 96-well plates overnight, then added Edu solution (25 μM) to the wells for 1 day. We applied 4% formalin to fix the cells at RT for 2 h and permeabilized the cells with 0.5% TritonX-100 for 10 min. We then added 200 μL of Apollo reaction solution for 30 min to stain EdU and 200 μL of DAPI to stain the nuclei. Lastly, we utilized a Nikon microscope (Nikon, Japan) to detect cell proliferation and DNA synthesis, reflected by blue and red signals, respectively.
Dual-luciferase reporter assay
We constructed WT/mut-circ-EPB41 or WT/mut-eIF5A 3'UTR fragments and inserted them downstream into a pMIR-REPORT plasmid luciferase reporter gene (H306, Obio Technology, Shanghai, China). We employed Lipofectamine 2000 to transfect the reporter plasmid into A549 cells and co-transfected the miR-486-3p mimic with the reporter gene into HEK293T cells. Afterwards, we employed the Dual Luciferase Reporter System Kit (E1910, Promega, Madison, WI, USA) to measure firefly and Renilla luciferase activity.
Animal studies
To detect the role of circ-EPB41 in a LC metastasis model, we intravenously injected 1×106 stable lentiviral-mediated circ-EPB41-silenced A549 cells (sh-circ-EPB41) or A549 NC cells into male nude mice (Chinese Science Academy, Shanghai, China) via the tail vein. After 1 month, we analyzed A549 cell metastasis by bioluminescence imaging following an intravenous luciferin injection (150 mg luciferin/kg body weight) into the tails. The numbers of metastatic foci in lung tissues were caculation according to the HE staining.
For xenograft assays, we injected 1×106 modified (circ-EPB41) or control (WT) A549 cells subcutaneously into the right flank of male nude mice. We calculated tumor volumes (length × width2 × 0.5) at the indicated time points and excised the tumors 4 weeks after injection.
We maintained all mice and handled them following procedures approved by the Animal Care Committee of Shanghai University of Medicine and Health Sciences.
Immunohistochemistry
We fixed tumor tissue samples in 10% formalin and embedded them in paraffin. We stained sections (5-μm thick) with Ki67 to evaluate proliferation. We examined sections with an Axiophot light microscope and imaged them by digital camera.
Statistical analyses
We assessed differences between any two groups by paired/unpaired two-tailed t-tests. We used Pearson’s correlation test to detect correlations between groups. We expressed data as means ± SD. A P-value <0.05 was regarded as significant. We performed statistical analysis using the GraphPad Prism package (GraphPad Inc., San Diego, CA, USA).