Clinical samples
Fresh frozen PC tissues and their adjacent normal tissues were obtained from the Pancreas Biobank of the First Affiliated Hospital of Nanjing Medical University, which passed ISO9001:2015 certification in 2018. The samples were taken within 10 mins after tumor excision and immediately stored at −80°C until application in the experiments. Written informed consent was acquired from all patients, and this study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University.
Cell culture
Human PC cell lines (Mia-PaCa-2, BxPC-3 and ASPC-1) and human embryonic kidney cells (HEK-293T cells) were purchased from American Type Culture Collection (ATCC, Manassas, USA), and the normal human pancreatic duct epithelial cell line (HPNE) was obtained from Pancreas Institute, Nanjing Medical University. All cells were incubated at 37°C in humidified air with 5% CO2 and maintained in Roswell Park Memorial Institute 1640 (RPMI1640, Gibco, CA, USA) or Dulbecco’s modified Eagle medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, CA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin, with or without 2.5% horse serum (Gibco, CA, USA).
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from cells and tissues using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The NanoDrop ND2000 (Thermo Scientific Inc., USA) was used to determine the purity and quantify the concentration of RNA. The First Strand cDNA Synthesis Kit (TakaRa, China) was used to perform reverse transcription. Primers used for real-time quantitative polymerase chain reaction (qRT-PCR) were obtained from TsingKe (Nanjing, China). According to the manufacturer’s instructions, qRT-PCR was performed using the SYBR Prime-Script RT-PCR kit (Roche, Germany) in an ABI LightCycler 96 fluorescent quantitative PCR system (Thermo Scientific Inc., USA). The reaction started at 95°C for 5 mins, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s. ACTB was used as the internal control to normalize qRT-PCR results. Relative gene expression levels were measured using cycle threshold (CT) in the ΔΔCT calculation. All oligonucleotide sequences are listed in Supplementary Table 1.
Construction of cell lines stably expressing KCNK15-AS1 and ACTR3B
For stable overexpression of KCNK15-AS1, full-length KCNK15-AS1 cDNA was synthesized by TsingKe (Nanjing, China) and cloned into the pCDH retroviral vector, named KCNK15-AS1-pCDH, which was confirmed by sequencing (TsingKe, Nanjing, China). The retroviral particle concentration and transfection steps were described in our previous research[7, 10, 11]. Positive clones were screened with puromycin for 2-3 weeks to establish KCNK15-AS1 stable expression cell lines and the corresponding negative control for further study. ACTR3B stable overexpression cells were established in the same way but using pHAGE vectors. For transient knockdown experiments, small interfering RNAs (siRNAs) were purchased from Ribobio Co. (Guangzhou, China).
Cellular phenotype in vitro
Cell proliferation, migration and invasion assay methods have been described in detail in our previous articles[7, 11]. For the colony formation assay, cells cultured to 90% confluence were harvested and seeded in 6-well plates at a dose of 200 cells per well. Fourteen days later, the cells were rinsed in phosphate-buffered saline (PBS) and stained with 0.05% crystal violet for colony counting.
Cell cycle experiment
The cell cycle was exposed by flow cytometry assay using a Cell Cycle Analysis Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. After 48 h of transfection, the cells were harvested with PBS and then fixed in 75% alcohol overnight at -20°C. The fixed cells were washed three times with PBS and then stained with propidium iodide (PI) dyeing buffer, which contained 200 μg/ml RNase and 50 μg/ml PI, at room temperature for 30 mins in the dark. The cell cycle was examined by flow cytometry (LSR, BD Biosciences).
Western blot assay
Protein extracts from cells or immunoprecipitation samples were prepared using detergent-containing lysis buffer. Total protein (60 μg) was subjected to SDS-PAGE and transferred to a 0.45-μm PVDF membrane (Merck Millipore, Germany). Antibodies were as follows: anti-ACTR3B, anti-SDHA, anti-IMMT (Abcam, USA), and anti-GAPDH (Santa Cruz, USA). The bands were visualized with Immobilon Western Chemiluminescent HRP substrate (Merck Millipore, Germany) using the Fluorchem E System (ProteinSimple, CA, USA).
Immunohistochemistry (IHC)
Paraffin-embedded tissue sections were heated at 65°C for 3 h, dewaxed in xylene for 30 mins followed by a graded ethanol series (100%, 95%, 85%, 75% to water) and then subjected to antigen unmasking by autoclaving in citric acid retrieval buffer; endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 20 mins, then the slides were washed three times with PBS, incubated in 5% bovine serum albumin for 60 mins, and incubated with ACTR3B (1:100) antibody overnight in a humidified box. The slides were washed three times with PBS, incubated with HRP-conjugated secondary antibody for 120 mins, washed in PBS for 5 mins, developed using 3,3′-diaminobenzidine (DAB) reagent, stained with hematoxylin, rinsed in tap water for 5 mins, dehydrated in an ascending alcohol series (75%, 85%, 95%, 100%) and mounted. Images were captured using a light microscope.
RNA pulldown-coupled mass spectrometry analysis
In vitro biotin-labeled RNAs (KCNK15-AS1, its antisense RNA as unrelated control RNAs) were transcribed with Biotin RNA Labeling Mix and DIG RNA Labeling Kit (SP6/T7) (Roche) and purified with the RNeasy Mini Kit (QIAGEN). In advance, we mixed BxPC-3 cell protein with magnetic beads (Pierce™, Invitrogen) to remove the beads binding protein. Biotinylated RNAs were incubated with the remaining protein, and then streptavidin magnetic beads were added. Pulled down proteins were run on SDS-PAGE gels, and then gels were stained by silver staining. All pulldown protein samples were sent for mass spectrometry analysis (Shanghai Applied Protein Technology Co., Ltd.).
RNA immunoprecipitation assay
The RNA immunoprecipitation (RIP) assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore, USA) following the manufacturer's protocol. Briefly, BxPC-3 and Mia-PaCa-2 cells at 80-90% confluency were lysed in RIP lysis buffer, and 100 μl of cell extract was incubated with RIP buffer containing magnetic beads conjugated to human anti-ACTR3B or negative control normal IgG. The samples were incubated with proteinase K to digest proteins, and then the immunoprecipitated RNA was isolated and subjected to qRT-PCR.
Luciferase reporter assay
HEK-293T cells were cultured in 48-well plates and cotransfected with 50 ng of ACTR3B promoter luciferase reporter vector or pGL3-Basic control vector and 500 ng of KCNK15-AS1 vector using Lipofectamine 2000. Twenty-four hours after transfection, the luciferase activities were assayed using the Dual-Luciferase® Reporter Assay System (Promega, USA).
Tumorigenesis assay
The animal care and experimental protocols were approved by the Nanjing Medical University Experimental Animal Welfare Ethics Committee. Four-week-old male BALB/c nude mice were purchased from the Animal Center of Nanjing Medical University and maintained under pathogen-free conditions. Mice (six in each group) were injected subcutaneously with 0.2 ml of cell suspension containing 1 × 107 cells in the back flank. When a tumor was visible, it was measured every week, and its volume was calculated according to the formula volume = 0.5 × length × width^2.
Statistical analysis
The statistical analysis was performed by SPSS software (Version 22.0) and GraphPad Prism (version 5.0). The quantitative data are presented as the mean ± SD. The differences between the means of two samples were analyzed by Student's t-test. The correlations between KCNK15-AS1 expression and various clinicopathological or serological variables were analyzed by the Mann-Whitney U test. Survival distributions and overall survival rates were determined using the Kaplan-Meier method, and the significance of the differences between the survival rates was calculated by the log-rank test. All data are representative of at least three independent experiments, and a difference was considered statistically significant at*p < 0.05, **p < 0.01, or ***p < 0.001.