Patients
30 cases BCa tissues and matched adjacent tissue were acquired form Xiangya Changde hospital, during 2019. No patient received receive any preoperative therapy. Specimens were quick-freezed in liquid nitrogen after resection and stored in -80℃.This study was approved by the medical ethics committee of Xiangya Changde hospital, each patient has signed the informed consent.
Cell culture and transfection
Normal immortalized cell SV-HUC-1 and BCa cells 5637, T24, TCCSUP and UM-UC-3 were purchased from CELL RESEARCH (Shanghai, China). 5637, T24 were cultivated in RPMI1640. TCCSUP and UM-UC-3 were maintained in Minimum Eagle’s medium (MEM). SV-HUC-1 were cultivated in F-12K medium. All cultures blended with 10% FBS and 1% antibiotics. All the cells were maintained at 37℃ and 5% CO2.
siRNA oligonucleotides specifically targeted HYOU1 were synsthesis by GENERAL BIOL (Chuzhou, China) and transiently transfected in T24 and TCCSUP cells. The oligonucleotides information was listed in Table 1. Scramble oligonucleotides were set as a negative control. HYOU1 coding sequences were synsthesised by GENERAL BIOL (Chuzhou, China) and subcloned into the expression vector pcDNA3.1. Cells were transfected using Lipofectamine 2000 (Thermo Fisher, USA) according to the product protocol. After 48 h, cells were harvested .
Table 1
The sequence information of siRNAs
siRNA/Genes | Sequence (5’ 3’) |
HYOU1 siRNA-1 | GCACAAUAAACGGGUACUC |
HYOU1 siRNA-2 | GCACAAUAAACGGGUACUC |
FOXO1 siRNA | CCAGAUGCCUAUACAAACA |
scramble | CCGUAUCGUAAGCAGUACU |
RT-qPCR
The whole RNA was isolated using Trizol reagent (TIANGEN BIOTECEH, Chia), and then reversely transcribed into cDNA using the RevertAid™ First Strand cDNA Synthesis Kit (Thermofisher, USA) according to the product’s instructions. The expression of HYOU1 mRNA was examined by RT-qPCR. The primers sequences were following: forward 5’-CTTCCACATCAACTACGGCG-3’, reverse 5’-CTCTTCTGCGCTGTCCTCTA-3’. β-actin was used as a control for normalizing, the primers information were following: forward 5’-TTCCTTCCTGGGCATGGAGTC-3’, reverse 5’-TCTTCATTGTGCTGGGTGCC-3’. The PCR reaction was conducted on LightCycler480 system (Roche, Germany).
Western blot analysis
Proteins extrated from BC tissues and cells using RIPA reagent (Thermofisher, USA) were subjected to 10% SDS-PAGE electrophoresis and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with antibodies anainst HYOU1 (1:800, Proteintech), PI3K (1:800, Proteintech), p-PI3K (1:800, Proteintech), AKT (1:1000, Proteintech), p-AKT (Try705) (1:1000, Abcam), FOXO1 (1:1000, Abcam), p-FOXO1(1:500, Proteintech), at 4°C overnight. After rinsing with PBS for three times, the membranes were incubated with HRP-conjugated secondary antibody. the bloting was detected with the enhanced chemiluminescence reagent (Millipore, USA). β-acin was the internal control.
CCK-8 experiment
Cell counting kit-8 (CCK-8) was conducted to measure cell proliferation. Briefly, 5×103 cells per well were seeded in 96-well plate for 24 h. At the indicated time points (0h, 24h, 48h, 72h), each well was added 10 µl CCK-8 reagent (Beyotime Biothechnilogy, shanghai, China) and incubation at 37°C for 2 h. Then the optical density was measured at 450 nm with a microplate reader (Thermofisher, USA).
Wound healing
Cell migration was analyzed using wound healing assay. Each group cells were cultured in a 12-well plate. Cell wounds were scratched with a sterile pipette tip when the cells reached 90% -95% confluence. Afterwards, cells were rinsed twice using PBS and incubated with serum-free culture. Images were caputed at 0 and 24 h.
Transwell assay
500 µl serum-free culture containing 1×105 cells were plated in the upper chamber covered with Matrigel (Corning, USA), while the lower chamber was filled with medium containing 20% FBS. After incubating for 24 h, the penetrating cells of upper chambers were fixed by methanol and stained with crystal violet for 15 min. Then the images of the invasive cells were photographed.
Flow cytometry assay
Cell apoptosis was detected using the Annexin V-FITC/PI Apoptosis Detection Kit (BD, USA). Cell with 48h transfection were collected and resusupended in 500 µl Binding Buffer, then the cell suspension was added 5 µl Annexin V-FITC and 5 µl Propidium Iodide and incubated in the dark for 15 min. Finally the cell apoptosis was determind by flow cytometry (CytoFlex, Beckman, USA).
Luciferase reporter assay
In order to investigate whether HYOU1 was regulated by FOXO1 at transcriptional level, The fragment of HYOU1 promoter (nucleotide positions − 1818 ~ + 59) was synthesised and cloned to the firefly luciferase report vector pGL-3. Cells were transfected with the recombination reported plasmids, and further co-transfected with FOXO1 overexpression plasmids or FOXO1 siRNA. The promoter activity was measured with the Dual-Luciferase reporter assay Kit (Promega, Madison, USA) following the product’s protocol at 48h after transfection. Renilla luciferase was used to normalization for tanstection efficiency.
Statistical analysis
All the data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS 18.0 soft (SPSS Inc., IL, USA). Student’s t-test was used to assess the differences between two groups. All assays were repeated at least three times. P < 0.05 was considered statistically significant.