Tissue gathering and Ethics statement
A total of 17 cholangiocarcinoma patients analyzed in this study had undergone surgeries at the Second Affiliated Hospital of Nanjing Medical University (China). All tissue samples were instantly snap-frozen in liquid nitrogen until extraction of RNA. This study was approved by the Research Ethics Committee of Nanjing Medical University (Nanjing, Jiangsu, People’s Republic of China). Written informed consent was obtained from all participants.
RNA extraction and qRT-PCR analysis
All RNA was isolated from specimens or cultured cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with manufacturer’s instructions. Then, RNA (1 µg) was reverse transcribed to cDNA through a Reverse Transcription Kit (Takara, Dalian, China). For Real-time PCR analyses, we used SYBR Green (Takara, Dalian China). The expression data were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers sequences were listed in Supplementary Table S1.
CCA cell lines HuCCT1 and RBE were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cell lines were cultured in DMEM (Life Technologies Corporation Attn, Grand Island, USA) medium supplemented with 10% fetal bovine serum (FBS) (Sciencell, Carlsbad, CA), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Shanghai, China) in humidifed air at 37 °C/5% CO2.
LINC00152 and LRIG1 cDNA was synthesized by Generay (Shanghai, China) and Generay (Shanghai, China), respectively. And they were ligated into the expression vector pcDNA3.1 (Invitrogen). Scrambled negative control siRNA (si-NC) and LINC00152 siRNAs were purchased from Invitrogen (Invitrogen, CA, USA). EZH2 siRNAs was bought from Realgene (Nanjing, China). The shLINC00152 was cloned into pENTR™/U6 vector. Severally, lipofectamine2000 (Invitrogen) and X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) were used to transfect the siRNAs and plasmid into cells. Cells were harvested for analyses 48 h after transfections. All the siRNAs and shRNA sequences were presented in Supplementary Table S1.
Cell proliferation analysis
Cell viability was measured with CCK8 kit (Houston TX, USA) according to manufacturer’s instruction. In colony formation test, the transfected cells were placed in 6-well plates with suitable media containing 10% FBS. After 2 weeks, colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma). The numbers of visibly stained colonies were counted to determine the colony formation. Edu assays were performed using the Edu Cell Proliferation Assay Kit (Ribobio, Guangzhou, China) following the manufacturer’s instructions. Then, the percentages of Edu-positive cells were examined in the sample. Experiments were independently repeated three times in triplicate.
Cell migration assays
For migration assays, 3 × 104 transfected cells in media with 1% FBS were placed into the upper chamber of the insert (Millipore, Billerica, MA, USA), while the medium in lower chamber contained 10% FBS. After 24 h of incubation, cotton wools were used to remove the cells remaining on the upper chamber. The cells migrated through the membrane were fixed with methanol, stained with 0.1% crystal violet, and imaged and counted with an IX71 inverted microscope (Olympus, Tokyo, Japan). All wells were assessed three times.
Western blot assay and antibodies
Cells protein lysates were divided using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) transferred to 0.22 µm NC membranes (Sigma) and incubated with specific antibodies. The ECL chromogenic substrates were quantifed by densitometry (Quantity One software; Bio-Rad). An anti-GAPDH antibody was employed as a control. AntiEZH2 was from Millipore (Billerica, MA, USA) and Anti-LRIG1 was purchased from GeneTex (Irvine, USA).
Flow cytometric analysis
The HuCCT1 and RBE cells were harvested by trypsinization 48 h after transfection with si-NC or si-LINC00152. After double staining with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI), the cells were analyzed by flow cytometry (FACScan; BD Biosciences) equipped with CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA). Cells were stained with PI using the CycleTEST™ Plus DNA Reagent Kit (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by FACScan for cell cycle. The proportions of cells in G0/G1, S, and G2/ M phase were calculated.
In vivo tumor formation assays
Four-week-old Athymic male mice bought from the Animal Center of the Nanjing University (Nanjing, China) were kept under specific pathogen-free conditions. The HuCCT1 cells stably transfected with sh-LINC00152 or empty vector were harvested and washed with phosphate-buffered saline (PBS). The cells re-suspended at 2 × 107 cells/mL were subcutaneously xenografted into the ventral side of each BALB/c male nude mice. Then, the tumor volumes were calculated as V = 0.5 × D × d2 (V, volume; D, longitudinal diameter; and d, latitudinal diameter) every 3 days. 16 days after injection, the mice were asphyxiated by CO2 and the tumor weights were measured and analyzed. This study was strictly consistent with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was licensed by the Committee on the Ethics of Animal Experiments of Nanjing Medical University.
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) assays were conducted using EZ-CHIP KIT following the manufacturer’s instructions (Millipore, USA). EZH2 and H3 trimethyl Lys 27 (H3K27me3) antibodies were from Millipore and Abcam, respectively. The ChIP primer sequences were shown in Supplementary Table S1. Quantification of precipitated chromatin DNA was analyzed by qPCR. ChIP data were calculated as a proportion relative to the input DNA using the following equation: 2[Input Ct− Target Ct] × 0.1 × 100.
RNA immunoprecipitation assays
RNA immunoprecipitation (RIP) assays were conducted using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) in accordance with the manufacturer’s protocols. The EZH2 antibody used in RIP was obtained from Millipore (Billerica, MA, USA).
Whole transcriptome deep sequencing
Total RNA from LINC00152 knockdown and control HuCCT1 cells were separated and quantified. The concentration of each sample was measured with NanoDrop 2000 (Thermo Scientific, USA). The quality was evaluated with the Agilent2200 (Agilent, USA). The sequencing library of each RNA specimen was prepared using Ion Proton Total RNA-Seq Kit v2 (Life technologies, USA). Data of six samples are available in Supplementary Table S2 and Table S3.
Statistical analyses were performed with GraphPad Prism5 (GraphPad Software, La Jolla, USA). Student’s t-test, #2 test or Wilcoxon test were used to estimate the significance of the distinctions between groups. The dys-regulated genes from GEO and TCGA datasets were obtained by limma R package and edger R package, respectively. All resulting data were recounted as mean ± SD. Two-sided P-values were calculated, and less than 0.05 was considered for statistical significance.