Clinical tissue specimens
We collected 8 cancer tissue samples of ESCC.All samples were from the First Affiliated Hospital of Gannan Medical College,from January to December in 2019. After the surgical operation,tissue specimens were frozen in liquid nitrogen immediately.None of these patients had received radiation or chemotherapy.All patients were well informed, the processes were approved by Ethics Committee of The First Affiliated Hospital of Gannan Medical University, and written informed consent was obtained from each patient.
Two tissue microarrays were used to evaluate the expression of hsa_circ_0021727 by ISH.The tissue microarrays were purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China) and contained approximately 170 pairs of ESCC samples and 100 pairs of their para-carcinoma tissues. Patients were selected based on a clear pathological diagnosis of early stage (Stages IA-IIIA) ESCC. All patients’ follow-up records were collected from January 2006 to July 2015.
In order to screen out circRNAs that can be used as targets, we performed circRNA microarray hybridization and data analysis on the three pairs of tissue samples collected.Total RNA was digested with RNase R (Epicentre, USA) to remove linear RNAs and enrich circRNAs. Then, the enriched circRNAs were amplified and transcribed to fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar; USA). The labeled cRNAs were hybridized to an Arraystar Human circRNA Array (8x15K, Arraystar). After washing the slides,the arrays were scanned with an Agilent G2505C scanner.Agilent Feature Extraction software (version 22.214.171.124) was used to analyze the acquired array images.
CircRNA in situ hybridization (RNA-ISH)
All experimental steps will be performed according to the standard procedures of ISH. Tissue microarrays were deparaffinized, rehydrated through an ethanol gradient, and then treated with 20 µg/mL proteinase K (Roche Diagnostics, Indianapolis, IN). Then, it was fixed with formaldehyde (Thermo Scientific, Rockford, IL), rinsed twice with 0.13 M 1-methylimidazole, and finally fixed again with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Thermo Scientific). After blocking endogenous peroxidase in H2O2, prehybridization buffer was performed, and slides were finally hybridized with 200 nM didigoxigenin (DIG) LNA modifications.
Two pathologists blinded to clinical data scored tissue microarray staining independently using the following criteria.Two senior pathologists were blinded to patients’ outcome and assigned to evaluate the immunoreactivity independently. Intensity of immunostaining was scored as 0 (no immunostaining), 1 (weak immunostaining), 2 (moderate immunostaining), and 3 (strong immunostaining). The percentage of immunoreactive cells scoring was documented as 0 (none), 1(< 20%), 2 (20–50%), 3 (51–75%), and 4 (> 75%).Cutoff values for low and high expression groups were determined by using the degree × intensity staining rank. Low expression was defined as a final score < 6 and high expression was defined as a final score ≥ 6.
Human ESCC cell lines (TE-1 and KYSE-510) were purchased from American Type Culture Collection (USA). All cells were prepared in 10% fetal bovine serum(FBS; Gibco, USA) in RPMI 1640 medium (Gibco, USA). Culture dishes containing cells were incubated at 37°C in humidified air with 5% CO2.
Total RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from frozen tissues and cells using RNAiso Plus (Takara, Japan) according to the manufacturer's instructions. RNA level was measured by qPCR with the SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.cDNA was synthesized using PrimeScript RT Master Mix(Takara, China). qPCR was performed with TB Green Premix Ex Taq II (Takara, China) with the following thermal cycling program: 95°C for 30 s and 40 cycles at 95°C for 5 s, 60°C for 30 s, and a dissociation step.All primer sequences are recorded in Table S2.
CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).Short interfering RNA (siRNA) sequences were directly synthesized (GenePharma, Shanghai, China).Use Lipofectamine 3000 (Invitrogen, Carlsbad, CA) to transfect cells with designated lentiviral vectors according to the manufacturer’s instructions. All shRNA sequences were listed in Table S3A.
RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).Short interfering RNA (siRNA) sequences were directly synthesized (GenePharma, Shanghai, China).Use Lipofectamine 3000 (Invitrogen, Carlsbad, CA) to transfect cells with designated lentiviral vectors according to the manufacturer’s instructions. All shRNA sequences were listed in Table S3A.
Fluorescence in situ hybridization (FISH)
We purchased FISH probes designed by RiboBio (Guangzhou, China).Cy3-labelled probes and FAM-labelled probes were used as mark for hsa_circ_0021727 and miRNA-23b-5p(Table S2B).Nuclei were stained with 4',6-dimethyl-2-phenylindole (DAPI).The assay was performed according to the instructions of the FISH kit (Gene Pharma, China).Stained pictures were observed and acquired by confocal microscopy (Leica, Germany).
Cell proliferation, colony formation assays and 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay
[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) determination was used for the assessment of cell proliferation.The transfected cells were seeded in a 96-well plate, then 20 µl of 5mg/mL MTT solution (MTT Cell Proliferation and Cytotoxicity Assay Kit, BOSTER, China) was added to incubate for 4 hours, and finally 100 µL of dimethyl sulfoxide was added.The microplate reader measures the optical density at 490 nm.
Colony formation assays,divide the transfected cells into 6-well plates (1000 cells/well). After 2 weeks in culture, cells were fixed with 75% ethanol and finally stained with 0.2% crystal violet.The colony formation rate was determined by counting the number of stained colonies.
ethynyl-2’-deoxyuridine (EdU) immunofluorescence assay was performed with Cell-Light EdU DNA Cell Proliferation Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions.Treated esophageal squamous cell carcinoma cells were incubated with EDU for 3 hours.After fixation and permeabilization, anti-EdU reagents and DAPI were used for cell staining.Fluorescence microscopy was used to observe and obtain pictures.
Cell invasion, migration, wound healing assay
For the invasion assay, medium was added to the chamber after precoating the chamber membrane with 100 µL of Matrigel (BD Bioscience, San Jose, CA, USA). Then added 100 µL of serum-free medium to the transfected cells for use. Cells were added to the upper chamber, complete medium was added to the lower chamber, and sequentially incubated at 37°C in 5% CO2 for 24°h. Finally, fixed with 4% paraformaldehyde solution and stained with 0.1% crystal violet solution.We obtained pictures using an inverted fluorescence microscope using Olin and counted these cells.
Three-dimensional (3D) spheroid invasion assays were used to detect the invasive ability of tumor cells.Transfected cells were seeded in ultra-low attachment (ULA) round-bottom 96-well plates for 4 days, allowing them to form tumor spheroids. Then,added 100 µl of basement membrane matrix (BMM, Corning) to each well for centrifugation and incubated at 37°C for one hour to solidify. Finally, 100 µl of medium containing 10% FBS was added to each well and incubated at 37°C in 5% CO2. We obtained images with an inverted microscope.
For wound healing assay, transfected cells were added to a six-well plate culture in serum-free medium, followed by scraping with a pipette tip to form a cell monolayer. Representative images of cell migration were captured at 0 h and 24 h after linear wound formation. We assessed wound healing and obtained pictures by microscopy.
Western blot analysis
We added RIPA buffer to the transfected cells, then took the supernatant and added protease inhibitor (1%; ComWin Biotech, Beijing, China). Protein concentration was checked by BCA Kit (Biyotime Biotechnology). Equal amounts of protein were separated by SDS-PAGE (10% or 8%) and transferred to 0.45 µm PVDF membranes (Roche, Indianapolis, IN). After blocking with 5% nonfat milk for 1 hour, PVDF membranes were incubated with primary antibody overnight at 4°C. Membranes were washed a minimum of 3 times in TBST for 10 min each, followed by one hour incubation with goat anti-rabbit or goat anti-mouse secondary antibodies. Finally, the membrane was washed 3 times with TBST again.Protein bands were detected with
SuperSignal West Femto Agent (Millipore) and visualized with the Chemical Mp Imaging System (Bio-Rad).The antibodies are listed in Table S4.
RNA pull-down assay
A hsa_circ_0021727 biotin-conjugated probe and oligo probe(Table S3B) were designed by RiboBio (Guangzhou, China).The treated cells were added to the lysate and incubated with streptavidin magnetic beads (Life Technologies, USA) for 2 hoursThe cell lysates were incubated with probe-coated beads at 4°C overnight. The beads were washed five times repeatedly, then the bound miRNAs in the pull-down material were extracted using Trizol reagent and analyzed by qRT-PCR analysis.
Luciferase reporter assay
After designing and synthesizing wild-type and mutant vectors, ESCC cells were co-transfected with other lentiviral vectors.Cell lysates were added after 48 hours, and luciferase activity was analyzed by a dual-luciferase reporter gene assay system (Promega, USA).
Total RNA of ESCC cells was extracted in accordance with the manual of TRIzol®reagent (Invitrogen, Shanghai, China). The libraries were then constructed using TruSeq Stranded Total RNA with Ribo-Zero Gold according to the manufacturer's instructions.These libraries were sequenced on the Illumina sequencing platform HiSeqTM 2500(Aksomics,Shanghai, China).
All male BALB/c nude mice (20 ± 2 g) were purchased from Guangdong Medical Laboratory Animal Center (MLAC). All animal experiments were performed in accordance with the principles and procedures outlined in the Gannan Medical University Guide for the Care. Approval was obtained from the the First Affiliated Hospital of Gannan Medical University Animal Ethics Committee.
To observe tumor growth, the transfected cells were injected subcutaneously into the left side of the axilla. Tumor growth was evaluated by detecting the volumes of the xenografts once a week. Specifically, the formula for calculating the volumes was defined: (volume) = 1/2 x (long axis) x (short axis).
To establish a lung metastasis model, cancer cells stably expressing firefly luciferase were injected into 4-week-old BALB/c nude mice from the tail vein.Six weeks after injection, bioluminescent pictures of tumor lung metastases were obtained using an in vivo imaging system. The mice were sacrificed, and the lung tissue of nude mice was taken out to record the number of lung metastases, and histological sections and HE staining were performed.
SPSS (version 22.0) and GraphPad Prism 9.1 (GraphPad Software Inc., CA, USA) were used for statistical analysis. Student’s t test and/or chi-squared test were statistically employed for data analysis. Results were indicated as mean ± SD. P < 0.05 was considered significant.