Ethics statement
We collected 30 fresh PC tissues and paired adjacent tissues after obtaining informed consent from patients at the Affiliated Zhongda Hospital, Southeast University, Nanjing. We snap-froze samples in liquid nitrogen and stored them at -80°C before RNA extraction and fluorescence in situ hybridization (FISH). The Ethics Committee of the Affiliated Zhongda Hospital of Southeast University (Nanjing, China) approved this research, and the study was carried out in accordance with The Code of Ethics of the World Medical Association. The ethic approval code was 2016ZDSYLL027.0.
Fluorescence in situ hybridization (FISH)
We prepared particular probes to hsa_circ_0074298 (Dig-5′-GCCTCAACCACGGAGTTCCTTTTGCTCGG-3′-Dig) by Geneseed Biotech (Guangzhou, China). We detected signals using fluorescein isothiocyanate (FITC)-conjugated anti-biotin antibodies and Cy3-conjugated anti-digoxin antibody (Jackson ImmunoResearch, West Grove, PA, USA). We counterstained nuclei with 4,6-diamidino-2-phenylindole, and obtained images using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Cellculture
We obtained PC cell lines (PANC-1, SW1990, AsPC-1, and BxPC-3) as well as normal human pancreatic duct epithelial (HPDE) cells from the American Type Culture Collection. The culture medium consisted of 10% fetal bovine serum (FBS; Invitrogen Life Technologies, Carlsbad, CA, USA) and penicillin in Dulbecco’s modified Eagle’s medium. We cultured the cells in an incubator at 37°C with 5% CO2.
Bioinformatics analyses
We predicted circRNA/miRNA target genes using Circular RNA Interactome, and predicted the interactive relationships between miR-519 and SMOC2 using TargetScanHuman.
Celltransfection
The cDNA oligonucleotides specifically targeting hsa_circ_0074298 (sh-circ0074298), miR-519 inhibitor, and SMOC2 overexpression vector were synthesized by GenePharma (Shanghai, China). Both sh-circ0074298 and SMOC2 overexpression vectors were inserted into pcDNA3.1. Expression of hsa_circ_0074298, miR-519, and SMOC2 were then monitored using RT-qPCR and western blotting after transfection of PC cells for 48 h.
Lentivirus transfection assay
The full-length cDNA cloning vector was purchased from GenePharma (Shanghai, China) and used for the amplification template. The SMOC2 full-length cDNA was amplified with NheI and BamH I restriction sites using KOD-PLUS Neo polymerase. Then, the PCR-amplified cDNA was cloned into the pcDNA3.1 vector using restriction enzymes and DNA ligase. The sh-circ0074298 sequence were also inserted into the pcDNA3.1 vector. The recombined vector was packed with VSV, REV, and GAG vectors and transfected into 293T cells using the Lipofectamine 2000 Reagent (Invitrogen) to obtain a virus supernatant. The virus supernatant was added into PANC-1 cells with polybrene. The transfected PANC-1 cells were cultured with puromycin and verified by western blotting or an RT-qPCR assay [15].
Transwell migration and invasion assay
We analyzed cell migration using Transwell chambers (8 µm, 24-well plates) (Corning, Corning, NY, USA) following standard procedures. Cells (1 × 104 cells) were added to the upper chamber and cultured for 24 h, then removed from the chamber upper surfaces using cotton swabs. We fixed the cells located on the lower surfaces with methanol for 10 min, followed by Crystal Violet staining. We imaged the stained cells and counted them in five fields, which were randomly selected. For the invasion experiments, we coated the chamber inserts with 200 mg/mL diluted Matrigel and dried them overnight under sterile conditions.
RNAextractionandRT-qPCR
We utilized TRIzol reagent (Invitrogen Life Technologies) to separate total RNA from cultured PC cells and tissues as previous research described [16]. The RNA concentrations were measured with a NanoDrop 2000 Spectrophotometer (Nano Drop Technologies, Wilmington, DE, U.S.A.) and 500 ng total RNA or miRNA was reverse transcribed into cDNA using the High Capacity RNA to cDNA Kit (Takara Biotechnology Co., Ltd., Dalian, China) or the microRNA Reverse Transcription kit (Takara Biotechnology Co., Ltd.). The primers used for RT-qPCR were: hsa_circ_0074298, forward: 5′-TTATTGATTATTACTGGCAAAAACG-3′, reverse: 5′-CTATGTGGTAGCGTTTAATGTTGGT-3′; miR-519, RT primer, 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCACTCT-3′; miR-519d, forward: 5′-CAAAGTGCCTCCCTTT-3′ and reverse: 5′-CAGTGCGTGTCGTGGAGT-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward: 5′-CCAAAATCAGATGGGGCAATGCTGG-3′ and reverse: 5′-TGATGGCATGGACTGTGGTCATTCA-3′; and U6, forward: 5′-CTCGCTTCGGCAGCACATA-3′ and reverse: 5′-AACGATTCACGAATTTGCTG-3′. The thermal cycle was: 30 s at 95˚C, 5 s for 40 cycles at 95˚C, and 35 s at 60˚C.
Cellproliferationassay
Following the CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan), we assessed cell growth of transfected cells in plates with 96 wells at 24, 48, and 72 h. We used a spectrophotometer (Thermo Scientific, Rockford, IL, USA) to detect the absorbance at 450 nm.
Colonyformationassay
We transferred BxPC-3 and PANC-1 cells into plates with 6 wells for 10 days. Then, we treated the colonies with 10% formaldehyde for 30 min, followed by staining for 5 min with 0.5% Crystal Violet. Image-Pro Plus 6.0 (https://www.mediacy.com/imageproplus) was used for data analysis.
Cellcycleassay
A total of 2×105 cells/mL were diluted with RNase A in 75% ice-cold ethanol overnight, then stained with propidium iodide (PI; 50 mg/mL; MultiSciences Biotech, Hangzhou, China) in the dark for 30 min at 4ºC, followed by measuring with a flow cytometer (FACScan, BD Bioscience, San Jose, CA, USA).
Cellapoptosisassay
Flow cytometry binding buffer (100 μL) was added after harvested cells were washed twice using ice-cold buffer. The mixture containing 5 μL Annexin V/FICC and PI (BD, Franklin Lakes, NJ, USA) was used for staining of cells for 15 min in the dark, followed by 400 µL of binding buffer. We used a FACSCalibur flow cytometer (BD Biosciences) to analyze cell apoptosis.
Westernblotanalysis
We washed cells with precooled phosphate-buffered saline and then lysed them with cell lysis solution (RIPA). We detected the protein concentration via BCA (Thermo Fisher Scientific, Waltham, MA, USA). We then transferred proteins to a polyvinylidene difluoride membrane, and blocked them in TBST (25 mM Tris, 140 mM NaCl, and 0.1% Tween 20, pH 7.5) containing 5% skimmed milk and then incubated them for 2 h. We then incubated membranes in primary antibodies against SMOC2 and GAPDH (Abcam, Cambridge, MA, U.S.A.) at 4°C overnight. After washing (3× for 10 min) with TBST, we added secondary antibody and incubated them at room temperature for 1 h. The results were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Dual-luciferase reporter assay
We cloned the putative miR-519 binding site in the target gene, SMOC2, and wild-type (WT) or mutant (MUT) hsa_circ_0074298 3'-UTR into the psi-CHECK (Promega, Madison, WI, USA) vector downstream of the firefly luciferase 3'-UTR, or hsa_circ_0074298 as a primary luciferase signal with Renilla luciferase for the normalization signal, and named them as SMOC2-Wt/circ-0074298-Wt and SMOC2-Mut//circ-0074298-Mut. The psi-CHECK vector provided the Renilla luciferase signal for normalization to compensate for distinctions between harvested efficiencies and transfection. We performed transfection of HEK293 cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). We measured Renilla and firefly luciferase activities 1 day after transfection with the Dual-Luciferase Reporter Assay System (Promega, Mannheim, Germany) using a luminometer (Molecular Devices, San Jose, CA, USA), and analyzed relative Renilla luciferase activities following standard procedures.
Animal studies
For xenograft assays, we injected 1×106 modified (sh-circRNA, sh-circRNA + miR-519 inhibitor, or sh-circRNA + SMOC2) or control PANC-1 cells subcutaneously into male nude mice (Chinese Science Academy) in the right side. We measured tumor volumes (length × width2 × 0.5) at the indicated time points, and excised tumors 4 weeks after injection. Each group was comprised of six mice.
We maintained all mice and handled them according to the instructions of the Animal Ethics Committee of Affiliated Zhongda Hospital of Southeast University (Nanjing, China).
Statisticalanalysis
We conducted statistical analysis using SPSS version 13.0 software (IBM, New York, NY, USA). We utilized Student’s t-test to analyze the data, which are expressed as the mean ± SD. A value of p < 0.05 was regarded as statistically significant.