Tissue Specimens and Cell Culture
30 paired PC tissues and non-tumor adjacent tissues were collected from the Tianjin Baodi Hospital . Inclusion criteria: (1) there was no adjuvant therapy before surgery, including chemotherapy and radiotherapy. (2)PC pathology was confirmed by two experienced pathologists after surgery.(4) The patient could tolerate the operation without evident heart, brain and lung diseases. Exclusion criteria: (1) preoperative chemotherapy, radiotherapy and other related adjuvant therapies. (2) Patients could not tolerate surgery.This investigation was approved by the Ethics Committee of Tianjin Baodi Hospital. All patients agreed the protocol and signed the informed consent. This investigation was conducted in accordance with the Declaration of Helsinki.
Human pancreatic duct epithelial cell (H6c7) and human PC cell lines (Patu8988, SW1990, BxPC-3 and Panc02) were obtained from ATCC (Manassas, VA, USA). All cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Rockville,MD, USA) and incubated in a humidified atmosphere with 5% CO2 at 37°C.
Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted by TRIzol (Invitrogen,Carlsbad, CA, USA) reagent and cDNA was synthesized using reverse transcription reaction kit (TaKaRa, Komatsu, Japan) following the instruction. qRT-PCR was performed by SYBR Premix DimerEraser (TaKaRa, Komatsu, Japan) on a LightCycler 480 PCR System (Roche, Rotkreuz,Switzerland). Relative expression of RNAs was calculated by 2-ΔΔCT method and normalized to U6 or glyceraldehyde phosphate dehydrogenase (GAPDH). Primers are listed as below:CircCCT3 Forward primer: GGACCCAGGATGAAGAGGTT;CircCCT3 Reverse primer: CATTGGGTCCAAAAGCATCT; miR-613 Forward primer: GTGAGTGCGTTTCCAAGTGT; miR-613 Reverse primer: GGGTCCCTTCACACTTGGAA ; GAPDH: Forward primer: 5′-ACGCTGCATGTGTCCTTAG-3′; Reverse primer: 5′-GAGCCTCTTATAGCTGTTTG-3′; U6: Forward primer: 5′-CTCGCTTCGGCAGCACA-3′; Reverse primer: 5′-AACGCTTCACGAATTTGCGT-3′.
2×105 cells in each well were inoculated in 96-well plates. Medium was used as blank control. At post-hypoxiareoxygenation, indicated cells in per well were added 10 μL of CCK-8 (Beyotime Institute of Biotechnology, Beijing, China) solution, and kept at 37°C for 2 h. The OD value of 450 nm was detected. The final data was represented as cell viability.
For the apoptosis analysis, 1 × 106 cells were seeded in a 60 mm plate, treated with OE-CircCCT3 or CCT3 siRNA, and cultured for 24 h. After harvesting, 1 × 105 cells were suspended in a 1x binding buffer provided with the Annexin V-FITC Apoptosis Detection kit II (BD Bioscience, San Jose, CA, USA), then stained with FITC Annexin V(BD Bioscience) and PI (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence was detected with a BD Accuri C6 flow cytometer (BD Bioscience), and the data were analyzed with the BD Accuri C6 software (BD Bioscience).
Cells were plated into six-well plates and grown to nearly 90% confluence. The same size scratch was made through the cell monolayer using a 200 μL disposable pipette tip. After washing with PBS, fresh culture medium was added, and the cells were incubated at 37 °C under an atmosphere with 5% CO2. Wound closure was imaged at 0 and 48 h.
Circular RNA Interactome software (https://circinterac tome.nia.nih.gov) was used to predict CircCCT3-miRNA interactions, while the mRNA targets of miR-613 were predicted by TargetScan software (http://www.targetscan.org)
Luciferase Reporter Assay
First, a luciferase reporter vector (pGL3-Firefly_Luciferase-Renilla-Luciferase) with VEGFA 3’ UTR or CircCCT3 was built, and the mutant vectors were constructed by GeneChem (Shanghai, PRChina). A luciferase vector and miR-613 mimic or mimic negative control were cotransfected into cells and incubated for 24 h. A dual luciferase reporter assay detection kit (Promega, WI, USA) was used to detect firefly and Renilla luciferase activities, which were measured on a Fluoroskan Ascent device (Thermo Fisher Scientific, USA).
Fluorescence in situ hybridization
Cy3-CircCCT3 and fluorescein isothiocyanate (FITC)-miR-613 probes were synthesized and obtained from RiboBio (Guangzhou,PR China). Hybridization assays were performed using a FISH Detection Kit (RiboBio, PR China). All images were captured by a confocal microscope (FV1000; Olympus, Tokyo, Japan).
RNA Immunoprecipitation (RIP) Assay
A RIP kit (Millipore, Billerica, MA, USA)was purchased for performing the RIP assay. PC cells were collected, and lysed by RIP lysis buffer. Then, the cell lysates were incubated with anti-Argonaute2 (Ago2) or immunoglobulin G (IgG; negative control) antibody in RIP buffer at 4°C overnight. Co-precipitated RNA was isolate dafter digestion with 150 μL protease K. The expression of CircCCT3 and miR-613 was detected.
Pulldown assay with biotinylated CircCCT3 and miR-613
Probe Panc02 cells (1×107) were obtained, lysed, and sonicated as indicated. To generate the probe-coated beads, C-1 magnetic beads (Life Technologies) were coincubated with the CircCCT3 probe for 2.5 h at 25℃. Then, the CircCCT3 probe or oligo probe was coincubated with cell lysates at 4℃ overnight. RNA was eluted and extracted by wash buffer and used for qRT-PCR. The CircCCT3 probe with biotinylation was synthesized and purchased from RiboBio (Guangzhou, PR China).The miR-613 was also tested using the same procedure.
Cells were lysed with precooled radio-immunoprecipitation assay lysis buffer supplemented with protease inhibitor (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min on ice. The supernatant was collected after centrifugation at 14,000 rpm at 4 °C for 20 min. The protein concentration was determined using a bicinchoninic acid (BCA) protein concentration determination kit (RTP7102; Real-Times Biotechnology Co.,Ltd., Beijing, China). The samples (20 μg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difuoride membranes. A GAPDH antibody was used as an internal reference. The membranes were washed with TBST and incubated with goat anti-mouse/rabbit antibody. Color development was performed using the chemiluminescence detection method, and images of protein bands were obtained for analyses. The primary and secondary antibodies were as follows: anti-GAPDH monoclonal antibody (Cell Signaling Technology, Danvers, MA,USA; 5174S, diluted 1/1000), anti-VEGFA (Abcam, Cambridge,UK;ab214424 diluted 1/1000), anti-VEGFR2 (Abcam,Cambridge,UK; ab134191, diluted 1/1000), goat anti-mouse immunoglobulin G (IgG) (ProteinTech; SA00001-1, diluted 1/3000), and goat anti-rabbit IgG (ProteinTech; SA00001-2, diluted 1/3000). Each western blotting was repeated at least three times.
Xenograft Tumor Model
Animal experiments were conducted with the permission of the animal care and use committee of Baodi Clinical College of Tianjin Medical University and performed in accordance with the guidelines of the National Animal Care and Ethics Institution. Panc02 cell line stably transfected with sh-CircCCT3 or sh-NC was established. Afterwards, Panc02 cells stably transfected with sh-CircCCT3 or sh-NC were subcutaneously inoculated into the right flank of BALB/c male nude mice (Model Animal Research Center Of Nanjing University, Nanjing, China) in sh-NC group and sh-CircCCT3 group. The volume of tumors was recorded every week . After inoculation for 28 days, these nude mice were euthanized and the tumors were dissected and weighed. The expression of CircCCT3 was detected by qRT-PCR.
All data were described as mean ± SD and analyzed with GraphPad Prism 7.0 software (GraphPad Prism, San Diego, CA, USA). One-way ANOVA with a Bonferroni correction or Student’s t-test was used to analyze the differences between groups. The association between CircCCT3 expression and clinicopathological features was evaluated using Chi-square test. Survival data were analyzed using Kaplan–Meier method and the log-rank test. The correlation of CircCCT3 expression between miR-613 expression was carried out using Spearman’s method. P<0.05 was regarded as statistical significance.