Frozen fresh paraffin-embedded CCA and para-cancerous tissues of 34 CCA patients were collected during January 2013 to October 2015 at the Second Hospital of Hebei Medical University. This research has been carried out in accordance with the World Medical Association Declaration of Helsinki. Informed written consent was obtained from each subject. This study was approval by the medical ethics committee of the Second Hospital of Hebei Medical University.
Cell culture and transfection
CCA cell lines, including RBE, TFK-1, HCCC9810, QBC939, HUCCT1 and normal human bile duct cell line (HiBEC), as well as HEK293T cells were all purchased from ATCC (Rockville, Maryland, USA). The cells were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum.
Establishment of Xenograft model in nude mice
Animal research has been approved by the Second Hospital of Hebei Medical University Animal Care and Use Committee (IACUC). To establish a xenograft tumor model, 5 mice were divided into each group. 200 μl of 1 × 106 cells were injected subcutaneously into the axilla of female Balb / C nude mice. After six weeks, the mice were sacrificed by CO2 asphyxiation and the subcutaneous tumor weight was measured.
Total RNA was extracted with triazole reagent (Invitrogen, Carlsbad, CA, USA). CDNA was generated using PrimeScriptRT kit (Thermo Scientific, Glen Brunie, MA, USA). qRT-PCR was performed using the TB Green real-time PCR kit (RR420A, Takara) according to the manufacturer's procedures. GAPDH serves as an internal reference for cytoplasmic gene expression. This expression is calculated by the 2-ΔCt method.
Cell proliferation assays
QBC939 and HUCCT1 cells were seeded at a density of 1 × 105 cells/well into 96-well Plates (Corning, New York, USA). Cell proliferation was analyzed using the cell counting kit-8 (CCK-8, Guangzhou, China) according to the manufacturer's instructions.
Tumor sphere assay
QBC939 and HUCCT1 cells were plated into 24-well plates (Corning, New York, USA) as a single-cell suspension and contained 2% B27 (Life Technology), 20ng / mL epidermal growth factor (Life Technology) and 20ng / mL bFGF was added into Dulbecco's modified Eagle's medium / F-12.
Proteins were extracted as previously described. Equal amounts of protein are was subjected to electrophoresis and subsequently transferred to a polyvinylidene fluoride membrane (Schwabacher Merck Micropore, Germany). Antibodies to CDK8, MST1, YAP1and TEAD1, p-YAP came from cell signaling technology. The antibody to β-actin was from Sigma. HRP-conjugated secondary antibodies are from Thermo.
PMir-RHPN1-AS1-WT or PMir-RHPN1-AS1-Mut were co-transfected into HEK293T cells by pmirGLO-Report luciferase vector. Transfection of miR-345-5p-mimics were performed using Lipofectamine 3000 according to the manufacturer’s instructions. After transfection for 48 hrs, the Renilla and Firefly luciferase activities could be determined through the Dual-Luciferase Reporter Assay System according to the manufacturer’s protocol.
In situ hybridization (ISH)
In situ hybridization was applied to examine the expression of RHPN1-AS1 as well as miR-345-5p on tissue sections according to the manufacturer’s procedures (BOSTER, Hubei, China) as previously described .
Immunochemistry (IHC) assay
The tumor sections were dried and dewaxed and were washed with 3% hydrogen peroxide (Sigma) diluted in methanol, and then incubated and heated in sodium phosphate buffer (pH 6.0). Samples were incubated with LATS1 (1:200, CST, Danvers, USA) primary antibodies overnight at 4 ° C, and then performed using a non-biotin horseradish peroxidase detection system according to the manufacturer's instructions.
The data are presented as mean ± SD, and we calculated the comparison by Student’s t test and x2 test for comparison of the differences between groups. The Kaplan-Meier method was applied to plot the survival curves. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, LaJolla, CA, USA). P <0.05 was recognized as having statistically significant difference.