1. Patients and specimens
The study was approved by the Ethic Committee of Third Hospital, Peking University. Written consent was obtained from all patients prior to surgery. A total of 71 HCC tissues and matched adjacent nontumor tissues were obtained from patients who underwent surgery in the Third Hospital, Peking University. None of the patients had received chemotherapy, radiotherapy, or other anticancer therapy before surgery. The patients were histologically confirmed by experienced pathologists. Paired tissue specimens (tumor and adjacent normal tissues) were collected from the patients, immediately frozen in liquid nitrogen and then stored at -80°C until use. Clinical information and follow-up data were collected and listed in Table 1.
2. CircRNAs microarray hybridization and data analysis
Total RNAs were digested with RNase R (20U/μl, Epicentre, USA) to remove linear RNAs and enrich circular RNAs. The enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Super RNA Labelling Kit; Arraystar, Rockville, MD, USA). The labelled cRNAs were hybridized onto the Human circRNA Array (8×15K, Arraystar). The slides were incubated for 17h at 65°C in a hybridization oven (Agilent, Santa Clara, CA, USA). CircRNAs differentially expressed with statistical significance between HCC and paired normal tissues (fold-change (FC)≥2 and p≤0.05) were identified through Volcano Plot filtering. Hierarchical clustering was performed to show the distinguishable expression pattern of circRNAs among samples.
3. Cell Culture
The human HCC cell lines (BEL-7402, BEL-7404, Huh7, Hep3B and HepG2) and normal hepatocyte cell HL-7702 were purchased from the Shanghai Institute of Cell Biology with a certificate of authenticity for each cell line. The samples were maintained in DMEM/RPMI1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 10 % heat-inactivated fetal calf serum, 2 mM L-glutamine, and 100 U/mL penicillin–streptomycin mixture (Gibco BRL, Grand Island, NY, USA) at 37 ℃ in 5 % CO2. After undergoing cell culture in our laboratory for several months, all cell lines used were evaluated for genetic identification prior to the experiments. Based on the appraisal reports provided by CoBioer Biosciences Co., Ltd., no mutations and contaminations were found in our cell lines.
4. Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
All primers used for qRT-PCR were designed in primer 5.0 software (Premier, Canada) and synthesized by Tsing Ke Biotech (Chengdu, China). The complementary DNAs (cDNAs) for circRNA, mRNA, or miRNA measurement were synthesized using random, oligo(dT)18 or stem-loop primers, respectively, using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed in triplicate using a Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific) on the CFX connect real-time system (Bio-Rad, Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as the intrinsic control for circRNA and mRNA, and U6 was used as the endogenous control for miRNA. Relative expression level of each gene was calculated using the 2−ΔΔCt method.
5. circEPS15-overexpressing vector construction and transduction
The circEPS15-overexpressing plasmid synthesized and cloned into the adenoviral vector GV138 (Genechem) with a Flag-tag. Full-length circEPS15 and its serial deletion performed using X-tremeGENE siRNA transfection reagent (Roche, Mannheim, Germany). For western blot analysis, cell proteins were prepared 72 h post transfection.
6. Wound-Healing Assay
The cells were seeded in 6-well plates and transfected following the manufacturer’s instructions. When the cell confluence reached approximately 80 % at 48 h posttransfection, cells were serum-starved for 12 h and then scratched within the confluent cell layer by using the fine end of a 100-lL pipette tip. Wound healing was observed at different time points within the scrape line. At this point, representative scrape lines were photographed by using an inverted Leica phase-contrast microscope (Leica DFC 300 FX) under 20X objective lens every 24 h. Image Processing and Analysis in Java was used to measure the wound-healing assays. Duplicate wells for each condition were examined, and each experiment was repeated three times.
7. Trans-well Assay
Cell invasion assay was performed in a trans-well chamber (24-well type, 8 mm pore size, Corning, NY, USA). BD Matrigel Basement Membrane Matrix was assessed according to the manufacturer’s recommended protocol. Then, 0.5 mL of serum-free medium was placed in the upper chamber; DMEM with 10 % FBS was added to the bottom chambers. Equal numbers (1X105) of cells were plated in the upper chambers of the quadruplicate wells and incubated at 37 ℃ for 72 h. Cells were then fixed with paraformaldehyde and stained with crystal violet, to visualize the nuclei. The results of three independent experiments were averaged. Image Processing and Analysis in Java was used to measure the trans-well assay.
8. Cell cycle and apoptosis assay
To assess the cell cycle and apoptosis, 3×105 treated cells were seeded into 6-well plates and cultured for 48h at 37°C. The cells for cell cycle analysis were digested using trypsin (Hyclone), washed twice with phosphate-buffered saline (PBS), and fixed in 70% ethanol overnight at 4°C. The cells were centrifuged at 500g for 5min, washed twice with cold PBS, and centrifuged. After treating with RNase A (0.1mg/ml) and propidium iodide (PI, 0.05mg/ml) purchased from 4A Biotech (Beijing, China) for 30min at 37°C, cell cycle analysis was performed through fluorescence-activated cell sorting flow cytometry (Beckman Coulter, Palo Alto, CA, USA). For the analysis of apoptosis, cells were trypsinised followed by two PBS washing steps. The cells were stained using the Annexin V/PI detection kit (4A Biotech, Beijing, China) for 5min at room temperature. The apoptotic cells were measured using flow cytometry (Beckman Coulter). All experiments were repeated at least three time.
9. Western blot analysis
For the detection of protein, cytoplasm and nuclear protein extracts were prepared from cells. The protein concentration of each sample was determined using a NanodropTM spectrophotometer (Thermo Scientific). Protein (100ug) from each sample was examined bySDS-PAGE(4% stacking and 10% separating gels) and then transferred overnight on to PVDF membranes(Millipore). The membranes were immunoblotted with the following: poly clonal anti-human TJP1 antibody (1:200, Abgent); CDH2 antibody (1:1000, Abcam); VIM antibody (1:1000, Cell Signaling Technology); EPS15 antibody (1:500, Abgent); GAPDH antibody (1:1000, Santa Cruz Biotechnology); Flag antibody (1:1000, Cell Signaling Technology) overnight. The blots were then incubated with peroxidase-conjugated goat anti-rabbit antibody or goatanti-mouse antibody (1:4000, Millipore) for 1 h. The PVDF membranes were subsequently subjected to immunoblotting analysis using an ECL immunoblotting kit according to the manufacturer’s recommended protocol (Beyotime Institute of Biotechnology, China).
10. IRES prediction
The IRESfinder[29] was used to identify the IRES sequence of circRNA. Each sequence of circRNA was evaluated by sliding window approach with window size of 174 nt and step size of 50 nt. The region with the highest score was considered to be the IRES sequence of circRNA.
11. Statistical analyses
All statistical analyses were performed using SPSS version 21.0 (IBM SPSS Inc., Chicago, IL, USA) and Prism version 5.0 (GraphPad Software, LaJolla, CA, USA) softwares. Categorical variables were expressed as a count or percentage and tested using χ2 or Fisher’s exact test, as appropriate. Continuous data are reported as mean± standard deviation (SD) and compared using Student’s t test, the one-way analysis of variance (ANOVA) test or Mann-Whitney test as appropriate. Correlations were calculated using Pearson’s correlation analysis. The cut-off value used to stratify patients into high and low expression groups was the median expression of target genes. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. All tests were 2-sided, and P<0.05 was considered statistically significant.