Patients tissue samples
The study was performed in accordance with the Declaration of Helsinki and the guidelines of the committee of the Human Ethics Committee of Xinhua Hospital. A total of 39 snap-frozen GBC tissues and paired adjacent normal tissues were acquired from patients diagnosed with GBC at Xinhua Hospital. All the enrolled patients of this study had never received preoperative therapy and tissue samples were collected and frozen in liquid nitrogen immediately after surgical resection. The patient information was shown in Table 1. All of participants signed informed consent before this study.
Table 1
The association between circTP63 expression and clinicopathological factors in GBC patients.
| | CircTP63 expression | |
Clinicopathological factors | The number of patients (n = 39) | Lower (n = 19) | Higher (n = 20) | P-value |
Age | | | | 0.839 |
≤ 60 | 24 | 12 | 12 | |
> 60 | 15 | 7 | 8 | |
Gender | | | | 0.798 |
Male | 11 | 5 | 6 | |
Female | 28 | 14 | 14 | |
Tumor size | | | | 0.894 |
< 5cm | 16 | 8 | 8 | |
≥ 5cm | 23 | 11 | 12 | |
Histological grade | | | | 0.429 |
well and moderately | 21 | 9 | 12 | |
Poorly and others | 18 | 10 | 8 | |
Lymph node metastasis | | | | 0.038* |
Negative | 18 | 12 | 6 | |
Positive | 21 | 7 | 14 | |
Adjacent organ invasion | | | | 0.511 |
No | 16 | 9 | 7 | |
Yes | 23 | 10 | 12 | |
TNM stage | | | | 0.264 |
I-II | 19 | 11 | 8 | |
III-IV | 20 | 8 | 12 | |
*P < 0.05.TNM, tumor-node-metastasis. |
Cell lines culture
Three human GBC cell lines (NOZ, GBC-SD, and SGC-996) and the human intrahepatic biliary epithelial cell line HIBEC used in the present study were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in DMEM (Gibco, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, USA) and 0.5% penicillin/streptomycin (Gibco, Invitrogen, USA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Cell transfection
Cell transfection was performed with Lipofectamine 2000 or 3000 Reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The siRNA sequences for transfection were purchased from Gene Pharma (Shanghai, China). The sequences for circTP63 was as follows: si-circTP63-1, 5’-GCCAACAGUGAGGGGCCGU-3’;si-circTP63-2,5’-CAACAGUGAGGGGCCGUGAGA-3’;siRNAcontrol(si-NC)5’-UUCUCCGAACGUGUCACGU-3’.MiR-217 mimic, miR-217 inhibitor and miR-NC were obtained from Gene Pharma (Shanghai, China).
CCK8 assays
Transfected NOZ and SGC-996 cells were seeded in 96-well plates (3×103 cells per well). After 2 h, cells were added with 10 µl of the CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) in each well of the plate. Then, cells were incubated for 2 h in the incubator. Finally, the absorbance was detected at 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
Flow cytometry analysis
Transfected cells were harvested, washed and then were fixed with 70% ethanol at -20°C overnight, Next, after RNase digestion, the cells were stained with 20 µg/ml Propidium iodide (PI; Beyotime, Shanghai, China) at 37°C for 30 min, and 100 µg/ml RNase A was subsequently added to the cells and incubated in a 4°C dark room for 30 min. Cell cycle was examined by flow cytometry using the FACS Calibur system (BD Biosciences, San Jose, CA, USA).
Cell migration and invasion assays
Cell migration or invasion assays were performed by transwell plates (BD Falcon, USA) and were coated without or without Matrigel in 24-well transwell chambers with 8 mm pore polycarbonate filters (Millipore, Billerica, MA, USA). 1 × 105 cells were cultured on the upper chamber in medium without serum, while the lower chamber was added with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, USA). After transfection at 48 hours, cells on the lower layer of the membrane were stained using 1% crystal violet for 30 min at room temperature. The cell number was counted by using an Olympus microscope.
Quantitative Real-Time PCR (QRT-PCR) analysis
Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen). The cDNA was synthesized using a Prime Script RT reagent kit (Takara, Japan). The mRNA expression was analyzed by using SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA) by an ABI 7900 Fast Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, USA). The GAPDH or U6 was used as reference. The primer sequences were as follow: the circTP63 forward: 5’-GCCCTCACTCCTACAACCATT-3’;reverse:5’-TTGTGTGCTGAGGAAGGTACT-3’;TheEZH2-forward:5’-TGCAGTTGCTTCAGTACCCATAAT-3’;EZH2-reverse:5’-ATCCCCGTGTACTTTCCCATCATAAT-3’;GAPDH-forward:5’-AAGGTGAAGGTCGGAGTCA-3’;GAPDH-reverse:5’-GGAAGATGGTGATGGGATTT-3’;U6-forward:5’-CTCGCTTCGGCAGCACA-3’;U6-reverse:5’-AACGCTTCACGAATTTGCGT-3’. The relative mRNA expression was calculated using the 2−ΔΔCt methods.
Western blotting assays
Total protein was extracted using a RIPA buffer (Beyotime, Beijing, China). An equal amount of total protein was separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes (Millipore, USA). The membrane was blocked with 5% non-fat milk and incubated with the primary antibody with E-cadherin (1:1000, Cell Signaling Technology, Houston, TX, USA). Vimentin (1:1000, Cell Signaling Technology, Houston, TX, USA), EZH2 (1:1000, Cell Signaling Technology, Houston, TX, USA) and GAPDH (1:2000, Abcam) overnight at 4°C. Next, the secondary antibodies were added for 1.5 hours and then each protein band was detected by the ECL detection system (Amersham Biosciences, Buckinghamshire, UK).
Luciferase reporter assays
The wide type (WT) circPVT1 or EZH2 3’-untranslated region (UTR) containing miR-217 targeting sequence and the mutated type (MUT) was amplified and cloned into the luciferase reporter plasmid psicheck-2 vector (Promega, Madison, WI). Cells were collected and lysed for luciferase detection 48 hours after transfection. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, USA). The relative luciferase activity was normalized against to the Renilla luciferase activity.
Biotin-coupled RNA pull down
The 3’end biotinylated miR-217 mimic or control RNA was designed and synthesized by Genepharm (Suzhou, China). NOZ were transfected with 50 nM of biotin-labeled miRNAs (Gene Create, Wuhan, China). Streptavidin-coupled Dynabeads (Invitrogen) were washed, resuspended in the buffer and then was added with the biotin-labeled miRNAs. After incubating at room temperature for 10 min, the coated beads were separated with a magnet for 2 min. The pulled-down RNA was extracted by Trizol reagent and followed by qRT-PCR analysis.
In vivo xenograft experiments
Xenograft experiments (n = 5/per group) were performed by using 3-week BALB/c nude mice. All animal protocols were approved by the Institutional Animal Care and Use Committee at Xinhua Hospital. 1 × 105 NOZ cells were transfected and were subcutaneously injected into the flank. The tumor volume and weight were evaluated every week, Tumor volume (mm3) = (length) × (width)2/2. After 4 weeks, mice were sacrificed and tissues processed for further histological analysis.
Statistical analysis
All statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc. La Jolla, USA). Statistical analysis was carried out using t test or Bonferroni Multiple Comparisons Test, A p value of less than 0.05 was considered statistically significant.