In this study, 32 intrahepatic tumor tissues were collected from CCA patients undergoing surgery in the Second Affiliated Hospital of Nanjing Medical University. All patients had not received treatment before. All operations were approved by the Research Ethics Committee of Nanjing Medical University of China and met the requirements.
Cell lines and culture conditions
Human CCA cell lines (RBE and HUCCT1) and normal biliary epithelial cells (HIBEpic) were obtained from the American Type Culture Collection (Manassas, VA) and cultured in In DMEM medium containing 10% fetal bovine serum (DMEM; Invitrogen) . The cells were cultured in an incubator at 37 ° C, 5% CO2 , and 90% relative humidity, and the cells were adherently grown. Fresh medium was replaced every 2-3 day(s) and passaged when the cell fusion reached 80%-90%.
RNA extraction and qPCR assays
We extracted total RNA from tissues or cultured cells by TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription of total RNA into cDNA by reverse transcription reagents (Takara, Dalian, China). Real-time PCR was analyzed by SYBR Premix ExTaq (Takara, Dalian, China). The results were normalized to GAPDH expression.Performing real-time PCR assays on the ABI 7500 system and collect data from the instrument. The primer sequences are listed in Table S1.
Transfection of CCA cells
Two separate AGAP2-AS1 (AGAP2-AS1 1 and 2), EZH2, CDKN1A, SP1,and scrambled negative control (NC) were purchased from Invitrogen and transfected into cells using Lipofectamine 2000 (Invitrogen, USA). shRNA is used for projects that require long-term experiments, such as clone formation and subcutaneous injection of animals.The interference sequences used are listed in Table S1. Plasmid vectors were from generay (Shanghai,China) and extracted using a DNA Midiprep kit (Qiagen, Hilden, Germany) and transfected into cells by Fugene reagent (Roche, Basel, Switzerland).
Cell proliferation assays
Cell proliferation assays were performed using Cell Counting Kit-8 (Promega). RBE and HUCCT1 cells transfected with si-AGAP2-AS1 for 24 hour(s), were seeded in 96-well plates and incubated at 37°C under 5% CO2. Relative cell growth was measured every 24 hour(s). After incubation with CCK8 solution for 2 hour(s), the absorbance was measured at 450nm.Colony formation assays were performed to monitor CCA cell clonality. RBE and HUCCT1 cells transfected with sh-AGAP2-AS1 were placed in a six-well plate and replaced with medium containing 10% FBS for approximately 14 day(s) with the medium replaced every 5 day(s) for colony formation assays. After 14 day(s), the medium was discarded and washed with PBS, fixed with methanol, stained with 0.1% crystal violet (Sigma-Aldrich).
Flow cytometric analysis.
RBE and HUCCT1 cells transfected with si-AGAP2-AS1 were harvested after 48 hours. After staining with FITC-Annexin V and propidium iodide (PI) using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences), cells were analyzed by flow cytometry (FACScan; BD Biosciences) by CellQuest software (BD Biosciences). The cells were divided into living cells, dead cells, early apoptotic cells and apoptotic cells, and the relative proportion of early apoptotic cells in each experiment was compared with that of the control group.
Male athymic BALB/c nude mice (4 weeks old) were maintained under pathogen free conditions. 1.5 × 106 cells were suspended in 0.1ml serum-free medium, mixed with 0.1ml ECMgel, injected subcutaneously into the back of nude mice, and re-injected with the same number of cells at the same site three days later.RBE cells stably transfected with sh-AGAP2-AS1 or an empty vector were injected subcutaneously into the axilla of the mouse, and the tumor volume was measured every 3 day(s). Eighteen days after the injection, the mice were sacrificed and the volume and mass of each subcutaneously growing tumor were examined. The formula for calculating tumor volume is: (L × W2) / 2, where L is the maximum length of the tumor and W is the maximum width of the tumor.Tumor tissues were used for qPCR analysis of AGAP2-AS1 levels, H&E staining and immunostaining of Ki-67 protein. The program was approved by the Animal Experimental Ethics Committee of Nanjing Medical University.
Subcellular fractionation location
Separation of nuclear and cytoplasmic fractions was performed using the PARIS kit (Life Technologies) according to the manufacturer's protocol.
Fluorescence in situ hybridization
RBE cells were seeded in 24-well plates, and when the cells accounted for 40% of the area, the medium was discarded. The cells were washed with PBS, fixed in 4% formaldehyde for 10 minute(s), and washed again with PBS. Treatment with PBS containing 0.5% Triton X-100 was followed by three washes with PBS. The pre-hybridization solution was then added, and the probe-containing hybridization solution was added overnight. After washing with 4X, 2X, and 1X SSC, DAPI staining was performed. The RNA FISH probe was designed and synthesized by Ribobio, (Guangzhou, China).
RNA immunoprecipitation (RIP) assay
We performed RIP experiments using the Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). HUCCT1 and RBE cells were lysed in complete RIP lysis buffer and whole cell extracts were incubated with beads. The beads were then washed with a wash buffer containing 0.1% SDS / 0.5 mg ml-1 protease. A qRT-PCR assay was performed corresponding to the purified RNA to detect the presence of AGAP2-AS1. Antibodies for RIP determination of EZH2 , Goods number:17-662.control IgG antibodies from Millipore (Billerica, MA, USA).
Chromatin immunoprecipitation (CHIP) assays
HUCCT1 and RBE cells were treated with formaldehyde and incubated for 10 minutes to generate DNA-protein crosslinks. The cell lysate was then sonicated to generate a 200-300 bp chromatin fragment and immunoprecipitated with EZH2 or H3K27me3 specific antibody, Goods number:17-662(Millipore, Billerica, MA, USA) or IgG as a control. After the precipitated chromatin was recovered, qRT-PCR analysis was performed.
Luciferase reporter assays
We used JASPAR (http://jaspar.gener0eg.net/) online database to predict potential transcription factor of AGAP2-AS1 promoter regions, and several SP1 binding motifs were identified. The AGAP2-AS1 promoter region (2000 bp) was inserted into a pGL3-basic vector (Promega, Madison, WI, USA). The Dual-Luciferase Assay Kit following manufacturer’s protocol.
Western blot analysis and antibodies
We isolated cell protein lysates using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). They were transferred to a 0.22 mm NC membrane (Sigma) and then incubated with specific antibodies. The ECL chromogenic substrate was used for quantification by densitometry (Quantity One software; Bio-Rad). Anti-CDKN1A was purchased from Abcam,AB109520, Dilution concentration 1x 5000 (Hong Kong, China).
Immunohistochemical (IHC) analysis
Xenograft tumor tissue samples were stained with H&E and immunostained for Ki67. Anti-Ki67 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). IHC staining results were professionally analyzed.
We used SPSS software for statistical analysis (SPSS, Inc., Chicago, IL, USA). Clinical pathology data were analyzed by chi-square exact testing. All data were expressed as the means ± SD (standard deviation), and analyzed using the Student’s t test to compare two groups of in vitro and in vivo data Results are reported as mean±standard deviation. Statistical significance was specified as P < 0.05 (*) or P < 0.01 (**).