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 approved this research, and the study was carried out in accordance with The Code of Ethics of the World Medical Association.
FISH
We prepared particular probes to hsa_circ_0074298 (Dig-5′-GCCTCAACCACGGAGTTCCTTTTGCTCGG-3′-Dig) by Geneseed Biotech (Guangzhou, China). We detected signals through fluorescein isothiocyanate (FITC)-conjugated anti-biotin antibodies and Cy3-conjugated anti-digoxin antibody (Jackson ImmunoResearch, West Grove, PA, US). We counterstained nuclei with 4,6-diamidino-2-phenylindole, and obtained images using Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Cell culture
We obtained PC cell lines (PANC-1, SW1990, AsPC-1, and BxPC-3) as well as normal human pancreatic duct epithelial cells, HPDE from the American Type Culture Collection. The culture medium consisted of fetal bovine serum (FBS; Invitrogen Life Technologies, Carlsbad, USA) of 10% in Dulbecco’s Modified Eagle’s Medium, as well as penicillin. We cultured cells in an incubator at 37 °C with CO2 of 5%.
Bioinformatics analyses
We predicted circRNA/miRNA target genes using Circular RNA Interactome, and predicted the interactive relations between miR-519 and SMOC2 through TargetScanHuman.
Cell transfection
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 pGPH1/Neo. 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.
Transwell migration assay
We analyzed cell migration via Transwell chambers (Corning, Corning, NY, USA) following standard procedures. After incubation for 1 day, we removed cells on the chamber upper surfaces by cotton swabs. We fixed cells locating on the lower surfaces with methanol for ten minutes, followed by Crystal Violet staining. We imaged stained cells and counted them in five fields that randomly selected. In invasion experiments, we coated chamber inserts with 200 mg/mL Matrigel and dried them overnight under sterile conditions.
RNA extraction and RT-qPCR
We utilized TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) to separate total RNA from cultured PC cells and tissues. Using the Reverse Transcription Kit (Takara Biotechnology Co., Ltd., Dalian, China), we reverse-transcribed total RNA to cDNA. The primers used for RT-qPCR are: 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.
Cell proliferation assay
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.
Colony formation assay
We transferred BxPC-3 and PANC-1 cells into plates with 6 wells for ten days. Then, we treated the colonies with 10% formaldehyde for half of an hour, 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.
Cell cycle assay
A total of 2×105 cells/mL were diluted with RNase A in 75% ice-cold ethanol overnight, then we stained cells 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).
Cell apoptosis assay
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) of 5 μL was used for staining of cells for 15 min in the dark, followed by another 400 µL binding buffer. We used FACSCalibur flow cytometer (BD Biosciences) to analyze cell apoptosis.
Western blot analysis
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 two hours. 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 under room temperature for one hour. We analyzed results using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Dual-luciferase reporter assay
We cloned 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 through 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) via 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) right side. We measured tumor volumes (length × width2 × 0.5) at the indicated time points, and excised tumors 4 weeks after injection.
We maintained all mice and handled them according to the instructions of the Animal Ethics Committee of Affiliated XXXX Hospital, XXXX University Medical School.
Statistical analysis
We conducted statistical analysis via SPSS statistical software (SPSS, Chicago, IL, USA). We utilized Student’s t-test to analyze the data, which are denoted by the mean ± SD. p < 0.05 was regarded as statistical significance.