Clinical patient samples
Ninety pairs of HCC tumor and adjacent tissues were collected from patients admitted to the Department of Hepatobiliary Surgery, Yantai Mountain Hospital. All specimens were immediately frozen and stored in liquid nitrogen for further use. The detailed clinicopathological characteristics are described in Table 1. Inclusion criteria for HCC patients were curative hepatectomy performed between 2018 and 2019, diagnosed as HCC by two senior pathologists and no adjunctive treatment prior to the surgery. The exclusion criteria were other tumors, and incomplete clinical or prognostic data. For sampling, HCC tumor tissue was excised surgically while paired adjacent non-cancerous tissue was resected at the tumor over 3 cm away from the edge of the cancerous tissue. All samples were collected immediately after removal from the patients and snap-frozen in liquid nitrogen, followed stored at -80 °C before further experiments. The study was approved by the ethical committee of the Department of Hepatobiliary Surgery, Yantai Mountain Hospital. Written informed consent was obtained from all patients.
Cell culture and transfection
One human normal hepatic epithelial cell line THLE-21 and six HCC cell lines (Hep3B, Huh7, MHCC97L, SK-hep1, SNU-387, SMMC7721) were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA) in a humidified 37 °C incubator with 5% CO2. All cell lines used in our study have been authenticated using STR profiling. For circTP63 or ZBTB18 overexpression, the full-length sequence of circTP63 or ZBTB18 was cloned into pcDNA3.1 (Invitrogen, CA, USA) plasmid to generate pcDNA3.1-circTP63 or pcDNA3.1-ZBTB18. All small interfering RNAs (siRNAs), miR-155-5p mimics, inhibitor, and their NC negative controls were obtained from GeneCopoeia (Guangdong, China). Cell transfection was conducted with Lipofectamine 2000 Reagent (Invitrogen, CA, USA) as the manufacturer’s protocol. The siRNA sequences for transfection as follows:
si-circTP63#1, 5′- GCCAACAGUGAGGGGCCGU -3′;
si-circTP63#2, 5′- CAACAGUGAGGGGCCGUGAGA -3′;
siRNA control (si-NC) siRNAs 5′- UUCUCCGAACGUGUCACGU -3′.
Quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR)
TRIzol reagent (Invitrogen, USA) was used to extract total RNA from the collected tumor samples and cells following the protocols. The cDNA was synthesized using a PrimeScript RT reagent kit (Takara, Japan). Gene expression was analyzed with SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA) using an ABI 7900 Fast Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, USA), and GAPDH and U6 were served as an internal control to normalize mRNA and miRNA levels, respectively. The relative mRNA/miRNA expression was calculated using the 2-ΔΔCt method. The primers for qRT-PCR were as follows:
GAPDH F: 5′-AAGGTGAAGGTCGGAGTCA-3′;
U6 F: 5′-CTCGCTTCGGCAGCACA-3′;
circTP63 F: 5′-GCCCTCACTCCTACAACCATT-3′;
miR-155-5p F: 5′- GAGGGTTAATGCTAATCGTGATAGG -3′;
R: 5'‑ GCACAGAATCAACACGACTCACTAT ‑3';
StarBase 3.0 (http://starbase.sysu.edu.cn/) was used to predict the binding site of miR-155-5p within circTP63 and the targets of miR-155-5p as standard procedures.
Cell counting kit-8 (CCK-8) assay
Cells at a density of 3 × 103 per well were seeded into 96-well plates and incubated for 0, 24, 48 and 72 h. Next, 10 μl of CCK-8 solution (Dojindo, Japan) was added and incubated in the dark at 37°C for another 1 h. The absorbance was detected using the microplate reader (Synergy H4 Hybrid Reader, BioTek, USA) at a wavelength of 450 nm.
Colony formation assay
A total of 500 cells per well were plated into 6-well plates. After the cells were grown for about 10 days, cells were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, China) for 30 min, and colonies were photographed under a Nikon Inverted Research Microscope Eclipse Ti microscope and quantified with imageJ4.
Transwell assay was performed to evaluate cell migration and invasion. After transfection, 1×105 cells in 200 μL of FBS-free medium were seeded in the top chamber with a porous membrane with Matrigel solution (BD, USA) for invasion assay or non-coated membrane for migration assay, respectively. The bottom chamber was inserted into a 12-well filled with 800 μL complete medium containing 10 % FBS. After 24 h of culturing at 37 °C, cells were removed from the upper surface of the membrane with cotton swabs, and cells on the lower surface of the chamber were fixed with 4% formaldehyde and stained with crystal violet (Beyotime, China). Five random fields per well were photographed and calculated using a Nikon Inverted Research Microscope Eclipse Ti microscope.
Transfected cells (1 × 106 cells/well) were plated in 6-well plates. After treatment, cells were collected by centrifugation at 1500 rpm for 5 min and then incubated with 5 μL of FITC-conjugated Annexin V and 5 μL of PI for 20 min in the dark at 4 °C. The stained cells were detected by the BD FACS Aria II flow cytometer (BD Biosciences, USA).
In vivo xenograft experiments
Six-week BALB/c nude female mice were used to perform xenograft experiments (n = 6/per group). All animal protocols were approved by the Institutional Animal Care and Use Committee at the Department of Hepatobiliary Surgery, Yantai Mountain Hospital. 1 × 107 Hep3B cells transfected with the indicated siRNA using the in vivo transfection reagent, JetPEI (Polyplus Transfection, Illkirk, France) were subcutaneously injected into the flank. Mice were observed daily, and caliper measurements began once tumors became visible. The tumor volume was evaluated every seven days via calipers, which were calculated using the following formula: Tumor volume (mm3) = (height) × (width)2/2. After 35 days, mice were sacrificed, and tumors were dissected and weighed. Tumor tissues were collected and snap frozen in liquid nitrogen and stored at -80 °C for following analyses.
Nuclear cytoplasmic fractionation
The cellular fraction was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher, USA) as the manufacturer's protocol. 5×106 cells were re-suspended in buffer C (20 mM Tris-HCl pH 7.5, 75 mM NaCl, 5 mM MgCl2, 0.5% p/w sodium deoxycholate, 0.2% Triton, 1 mM DTT, 0.5% glycerol) supplemented with protease inhibitor cocktail (Sigma, USA) and 1 U/μL RNase inhibitor (Thermo Scientific, USA). After centrifugation, cytoplasmic lysate supernatants were carefully harvested. Then pelleted nuclei were washed extensively with PBS. Pelleted nuclei were resuspended in buffer N (10 mM Tris-HCl pH 8, 25 mM NaCl, 5 mM MgCl2, 1% p/w sodium deoxycholate, 1% Triton, 0.2% SDS, 1mM DTT) supplemented with protease inhibitors and RNase inhibitors. After sonication, RNA from each portion was isolated for further analysis.
Ribonuclease R (RNase R) treatment
3 μg total RNAs were incubated at 37°C in 20 μL reactions containing 2 μL of 10× RNase R Buffer (0.2 M Tris-HCl (pH 8.0), 1 mM MgCl2 and 1 M KCl, NaCl or LiCl) and 1 μL of RNase R (20 U/μL, Epicentre, USA) to remove linear RNAs and enrich circRNAs at 37 °C for 45 min. Controls were also conducted but the water was added instead of the RNase R enzyme. After RNase R treatment, the reactions were incubated at 70 °C for 10 min to deactivate the RNase R. The treated RNAs were used for qRT-PCR.
Luciferase reporter assays
A total number of 3 × 104 cells were plated in 24-well plates the day before transfection. Then cells were transfected with the wild-type or mutant circTP63/ZBTB18 reporter plasmids using Lipofectamine 2000 (Invitrogen, USA) as the manufacturer’s instructions. After 48 h of transfection, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, USA).
circTP63 and negative control Oligo were biotinylated by GenePharma Company (Shanghai, China). Next, they were transfected into HCC cells at 4 °C for 48 h. Cells were collected and incubated with Streptavidin-coupled magnetic beads (Invitrogen, USA) at 25 °C for 2 h. After cells were washed and eluted with buffer, the bound RNAs were quantified and analyzed by qRT-PCR.
Western blot analysis
After treatment, cells were lysed in RIPA lysis buffer (Beyotime, China). Total protein concentration was subjected to the BCA Protein Assay (Beyotime, China). Next, proteins were separated on SDS-PAGE and transferred onto PVDF membranes (Millipore, USA), followed by blockade with 5% non-fat milk for 1 h at room temperature, and then incubated with corresponding primary antibodies (ZBTB18, ab118471, dilution 1:1000; GAPDH, ab181602, dilution 1:10000) at 4 °C overnight. Then, the membranes were incubated with corresponding secondary antibodies. All these antibodies were purchased from Abcam (Cambridge, UK). An Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, USA) was used for protein bands detection. The protein bands were quantified with the ImageJ4.