Animals
We used BALB/c nude mice with four weeks old weighing 15–20 g (SLARC, Shanghai, China) in this study. Ethics Committee in Shanghai University of Medicine and Health Sciences approved all animal experiments.
Patients and samples
In total, we enrolled 90 pairs of BC tissues and adjacent normal tissues of BC patients who were recruited in Huashan Hospital, Fudan University School of Medicine between January 2007 and January 2013. No patients received preoperative local or systemic treatment prior to specimen collection. We acquired informed consent from every patient or his/her relative(s). The Board and Ethics Committee in Huashan Hospital and Fudan University School of Medicine approved this project. We directly deposited collected tissues in liquid nitrogen for further usage.
Strand-specific high-throughput RNA-Seq library construction
We extracted total RNA from paired BC and adjacent noncancerous tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). We subjected nearly 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 erase ribosomal RNA, but retain other RNA classes such as non-coding RNAs and mRNAs. We treated the RNAs with 40 U of RNase R (Epicenter) at 37 °C for three hours, followed by TRIzol purification. We prepared RNA-seq libraries by the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) and subjected them to deep sequencing through Illumina HiSeq 4000 at Aksomics, Inc. (Shanghai, China). For miRNA and mRNA analyses, T24 cells transfected with siRNA against hsa_circ_0001944 or negative control (NC) vector were used for high-throughput RNA-Seq of miRNAs as previously mentioned.
Cell culture and transfection
We purchased SV-HUC-1 cells as well as the BC cell lines 5637, UM-UC-3, T24, and RT-4 from Type Culture Collection in Chinese Academy of Sciences (Shanghai, China) and cultured them in DMEM (Gibco, Grand Island, NY, USA) supplemented with fetal bovine serum (FBS) of 10% under 37°C in a humidified incubator with CO2 of 5%.
We transfected small interfering RNAs (siRNAs; si-hsa_circ_0001944 and si-circRNA), miR-548 mimics, miR-548 inhibitors, PROK2 overexpression vector, and their NCs into cultured T24 or UM-UC-3 cells via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the standard process. To further verify hsa_circ_0001944 effect using in vivo experiments, lentiviral stabilized hsa_circ_0001944 silenced (sh-circ0001944) T24 cells were constructed.
Bioinformatic analysis
We identified circRNA/miRNA target genes with CircularRNAInteractome. We predicted the interactive relationship between miR-548 and PROK2 using TargetScanHuman.
Fluorescence in situ hybridization (FISH)
We made specific probes against hsa_circ_0001944 (Dig-5′-GATACTTTATGAGGAGACTAAGGTGTCAGTATG-3′-Dig) with help from Geneseed Biotech (Guangzhou, China). We explored signals using 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). Afterwards, we acquired images with a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Total RNA isolation and quantitative reverse transcription (RT-q)PCR
We isolated total RNA from tumor tissues or cells with TRIzol reagent (Invitrogen) following standard protocol. We examined RNA sample purity and concentration spectrophotometrically through detecting absorbance at 260 nm, 280 nm, and 230 nm with a NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA). In particular, we deemed OD260/OD280 ratios ranging between 1.8–2.1 and OD260/OD230 ratios >1.8 as acceptable.
We reverse transcribed total RNA before RT-qPCR detection. We obtained primers specific for hsa_circ_0001944, miR-548, and PROK2 from GenePharma (Shanghai, China). We performed RT-qPCR with AB7300 thermo-recycler (Applied Biosystems, Carlsbad, CA, USA) with primers and the TaqMan Universal PCR Master Mix. We employed GAPDH as reference gene for circRNAs and mRNAs. We used U6 as an internal control for miRNA expression levels. We quantified gene expression via the 2−ΔΔCt method. The primers utilized to assay hsa_circ_0001944 expression included 5′-CTCTTTGACATCATAATAAAATACT-3′ forward, and 5′-GGCTGAGGCAGGAGAATAGCTTGGG-3′ reverse. The miR-548 primers were 5′-ATTGGAACGATACAGAGAAGATT-3′ forward and 5′-GGAACGCTTCACGAATTTG-3′ reverse. The PROK2 primers were 5′-GGGGATCCATGAGGAGCCTGTGCTGCGCCCCA-3′ forward, and 5′-GGGAATTCCTTTTGGGCTAAACAAATAAATCG-3′ reverse. The U6 primers were 5′-CTCGCTTCGGCAGCACA-3′ forward, and 5′-AACGCTTCACGAATTTGCGT-3′ reverse. The GAPDH primers were 5′-GCACCGTCAAGGCTGAGAAC-3′ forward, and 5′-GGATCTCGCTCCTGGAAGATG-3′ reverse.
Dual-luciferase reporter assays
We inserted binding sites for hsa_circ_0001944 and the PROK2 3′-UTR, termed hsa_circ_0001944-WT, hsa_circ_0001944-Mut, PROK2-3′UTR-WT, and PROK2-3′UTR-Mut into HindIII and KpnI sites of pGL3 promoter vector (Realgene, Nanjing, China) of the dual-luciferase reporter assay. We first plated cells into plates 24 wells, and transfected 80 ng of plasmid, 50 nM of miR-548 mimics, 5 ng of the Renilla luciferase vector pRL-SV40, and NC reagents into cells with lipofectamine 2000 (Invitrogen). We collected cells and measured them 2 d after transfection with Dual-Luciferase Assay (Promega, Madison, WI, USA), following standard instructions. We independently repeated all experiments three times.
Cell proliferation assay
We used the Cell Counting Kit-8 (CCK-8) assay to detect cell proliferation. We seeded transfected cells into plates 96 wells with density 5,000 cells/well in triplicate. We measured cell viability through CCK-8 system (Gibco) at 0, 1, 2, and 3 d after seeding, following standard procedures.
For colony formation assays, we seeded transfected cells into plates with six wells with density 2,000 cells/well and maintained them in DMEM containing FBS of 10% for ten days. We imaged and counted the resultant colonies after fixing and staining them.
5-Ethynyl-2′-deoxyuridine (EdU) assay
We used the EdU assay kit (RiboBio, Guangzhou, China) to investigate cell proliferation and DNA synthesis. We seeded 10,000 T24 or UMUC3 treated cells into plates with 96 wells overnight. The second day, we inserted EdU solution (25 μM) to plate and incubated the cells for 1 d. We then used formalin of 4% to fix cells at room temperature for two hours. We utilized 0.5% TritonX-100 to permeabilize cells for ten minutes, and added Apollo reaction solution (200 μL) to stain EdU and DAPI (200 μL) to stain nuclei for 30 min. Lastly, a Nikon microscope (Nikon, Tokyo, Japan) was used to measure cell proliferation and DNA synthesis, which were reflected by blue and red signals, respectively.
Transwell and wound healing assays
We suspended BC cells in 200 μL of serum-free medium and inserted them into upper chamber of Transwell plates with 8-μm pores (Corning Costar, Corning, NY, USA). We also placed 600 μL medium containing 20% FBS in lower chamber as chemoattractant. After incubation for 1 d, we fixed cells in the filter with methanol, stained them with crystal violet solution of 0.1%, and then counted them in three random fields of view (200×).
For wound healing assays, we seeded BC cells in 6-well plates. We created a linear scratch wound with a 20-μL pipette tip in the confluent monolayer of cells. After 2 d of incubation in medium without FBS, we observed and photographed wound closure under microscope. We conducted experiments in triplicate and repeated them three times.
Flow cytometric analysis of cell cycle progression
We fixed cells in ethanol of 70% overnight at 4°C, resuspended them in staining solution (Beyotime, Shanghai, China), and then incubated them for half of an hour under 4°C. We measured stained cells by flow cytometry (Beckman Coulter, Franklin Lakes, NJ, USA).
Animal studies
For the xenograft assays, we subcutaneously injected 1×106 modified (hsa_circ_0001944-silenced) or NC T24 cells into right side of each male nude mouse. We calculated tumor volumes (length × width2 × 0.5) at timepoints indicated and excised tumors 1 m after injection.
For metastasis analysis, we transfected 2×105 NC or hsa_circ_0001944-silenced T24 cells with luciferase expression vectors, and injected the cells intravenously into the tails of mice. After 30 d, T24 cell metastases were analyzed by bioluminescence imaging following an intravenous injection of luciferin (150 mg luciferin/kg body weight) into the tails.
Immunohistochemistry
We fixed tumor tissue samples in formalin of 10% and embedded them in paraffin. We stained sections (5-μm thick) with Ki67 to explore proliferation. We examined sections with an Axiophot light microscope and imaged them with digital camera.
Statistical analysis
We assessed differences among groups via paired/unpaired t-tests (two-tailed). We used Pearson’s correlation test to obtain associations between groups. Data are denoted by mean ± SEM. We considered P-values <0.05 as significant. We performed all statistics analysis with GraphPad Prism (GraphPad Inc., San Diego, CA, USA).