Cell lines and cell culture
Six human pancreatic cancer cell lines (BxPC-3, CFPAC-1, Colo-357, MiaPaCa-2, PANC-1, and SW1990) were purchased from the Shanghai Cell Bank (Shanghai, China). The normal human pancreatic ductal cell line, HPNE, was purchased from the American Type Culture Collection (ATCC, USA). Cell lines that stably overexpress YY1 (BxPC-3 and PANC-1), cell lines in which YY1 is knocked down, and control cell lines were prepared and cultured as previously described . Human pancreatic cancer cell lines were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). The HPNE cell line was cultured according to the recommendations of the ATCC.
Total RNA was extracted from different cell lines using Trizol reagent and reverse-transcribed into cDNA using the PrimeScript RT Master Mix according to the manufacturer’s instructions. For miRNA quantifications, miRNAs were extracted using the miRNeasy Mini Kit (Qiagen, China). Reverse-transcription was performed using the Mir-XTM miRNA First-Strand Synthesis Kit (Clontech, China) according to the manufacturer’s instructions. The mRNA expression levels were determined by qRT-PCR using the Step One Plus Real-Time PCR System (Applied Biosystems, USA) with the FastStart Universal SYBR Green Master according to the manufacturer’s instructions. The expression levels of the YY1 mRNAs and miR-30a were normalized to GAPDH and U6, respectively, using the 2−ΔΔCT method. Each qRT-PCR experiment was performed in triplicate and independently repeated three times.
Pancreatic cancer cells or xenograft tumor tissues were lysed in ice-cold lysis buffer containing the following reagents: 50 mM Tris–HCl (pH 7.4), 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and a complete protease inhibitor cocktail (1 tablet per 10 mL, Roche Diagnostics GmbH, Mannheim, Germany). The total protein concentration was determined using a BCA Protein Assay kit (Beyotime Biotechnology, China). Western blotting was performed using standard methods. The following dilutions were used for each antibody: anti-YY1 (ab109228, Abcam, 1:3000), anti-Beclin 1 (#669922, R&D, 1:1000), anti-ATG5 (ab108327, Abcam, 1:3000), anti-P62 (ab109012, Abcam, 1:10,000), anti-LC3A/B (#12741, Cell Signaling, 1:1000), and anti-GAPDH (SAM1003, Sun Shine Bio, 1:5000). Each blot was independently repeated three times.
All reagents for fixation, washing, and blocking were purchased from Beyotime (Beyotime Biotechnology, China). Pancreatic cancer cells were washed with PBS three times for 5 min each and then fixed for 10 min. They were permeabilized with 0.1% Triton X-100 in PBS for 10 min. They were then blocked for 1 h at room temperature, followed by incubation with anti-LC3A/B antibody (#12741, Cell Signaling, 1:1000) at 4 °C overnight. After washing with PBS extensively the following day, cells were incubated with Cy3-Labeled Goat Anti-Rabbit IgG (Beyotime Biotechnology, China) for 3 h at room temperature. Chromatin was stained with DAPI for 10 min, and cells were observed under a fluorescence microscope (Nikon, Eclipse 80i, Japan).
Transmission electron microscopy
Pancreatic cancer cells were washed with PBS twice and fixed in 2.5% glutaraldehyde in PBS at 4 °C overnight. Fixed cells were washed with PBS three times and post-fixed with 1% osmium acid in PBS for 3 h. After washing the cells three times with PBS, the samples were dehydrated in a graded ethanol series (30, 50, 70, 80, 90, 95, and 100%) for about 15–20 min for each incubation. Samples were then transferred to absolute acetone for 20 min. The samples were placed in a 1:1 mixture of absolute acetone, followed by incubation in a Spurr resin mixture for 1 h at room temperature. Samples were incubated in a 1:3 mixture of absolute acetone, followed by the final resin mixture for 3 h and a final Spurr resin mixture overnight. Finally, the specimens were placed in capsules containing embedding medium and heated to 70 °C for about 9 h. Sections 70 nm in thickness were generated using an ultramicrotome (Leica Ultracut R, Germany). Specimen sections were stained with uranyl acetate and lead citrate for 10 min each and observed using a JEM1230 (JEOL, Japan).
miR-30a reagents and transfection
A miR-30a mimic, miR-30a inhibitor, and negative control mimic were purchased from Ribobio (Guangzhou, China). Lentiviruses used to knockdown or overexpress human miR-30a, in addition to negative control lentiviruses, were purchased from Hanbio (Shanghai, China). All transfections were carried out according to the manufacturer’s instructions.
The luciferase assay was carried out as previously described . Briefly, dual-luciferase reporter plasmids were constructed by iGeneBio (Guangzhou, China). Transfection of all plasmids was performed using the Lipofectamine 3000 Reagent. Luciferase activity was measured 48 h after transfection using the Dual-Luciferase Reporter Assay Kit (Promega,USA). The assay was repeated at least three times in independent experiments.
ChIP assays were performed as previously described  using the EZ ChIP Kit (Merck Millipore, Germany) according to the manufacturer’s instructions. Briefly, cross-linked chromatin was sonicated into fragments ranging from 100 to 1000 base pairs. The chromatin was then immunoprecipitated using a ChIP-grade anti-YY1 antibody (ab12132, Abcam).
PANC-1 xenograft tumor model
All animal research procedures adhered to the guidelines of The Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by the Laboratory of Animal Care and Use Committee of Nanjing Medical University. Four-week-old female nude mice (BALB/cA-nu) were purchased from the Model Animal Research Center of Nanjing University. Twenty mice were randomly divided into four groups. To establish a xenograft subcutaneous implant tumor model, PANC-1-YY1-knockdown and vector control cells, PANC-1-miR-30a-knockdown and vector control cells (1.5 × 106 cells/100 μL per mouse) were injected subcutaneously. The mice were euthanized after 4 weeks, and tumor xenografts were resected.
All data were representative of at least three independent experiments. Statistical analysis was performed using the SPSS software (Version 22.0). Quantitative data are presented as mean ± SD. All statistical tests were two-tailed exact tests with a p < 0.05 considered significant.