Clinical samples
Total 42 fresh RB specimens and the paired adjacent normal retina specimens from the patients of RB were provided by the Ophthalmology Department of our hospital. None of the patients received anti-tumor treatment, chemotherapy and radiotherapy before surgery. In addition, the characteristics of RB patients were presented in Table 1. This research was permitted by the Ethics Committee of our hospital. All patients or their guardians have signed informed consent.
Cell cultures
Two RB cell lines (HXO-RB44 and Y79) and normal retinal vascular endothelial cell line (ACBRI-181) were supplied by American Type Culture Collection (ATCC, USA). The cell lines were cultured in RPMI‑1640 medium (Gibco, USA) containing 10% FBS and 1% penicillin/streptomycin. All cells were cultured in a humidified incubator with 5% CO2 at 37˚C.
Cell transfection assay
The pcDNA3.1-UCA1, UCA1 siRNA and their corresponding negative control (pcDNA3.1-Mock and UCA1 siRNA control) were obtained from Thermo Fisher, USA. LY294002 (PI3K inhibitor) was purchased from Sigma, USA. The pcDNA3.1-UCA1, pcDNA3.1-Mock, UCA1 siRNA and UCA1 siRNA control were separately transfected into HXO-RB44 and Y79 cells cells by Lipofectamine® 3000 Reagent (Invitrogen, USA). The transfected HXO-RB44 and Y79 cells were randomly divided into six group: BLANK group (no-treated group), pcDNA-UCA1 group (transfected with pcDNA3.1-UCA1), pcDNA-Mock group (transfected with pcDNA3.1-Mock), si-UCA1 group (transfected with UCA1 siRNA), si-NC group (transfected with UCA1 siRNA control) and pcDNA-UCA1 + LY294002 group (transfected with pcDNA-UCA1 and treated with 50 μM LY294002). Finally, all cells were cultured in 37°C for 48 hours.
Cell counting kit-8 assay
The cell proliferation abilities of HXO-RB44 and Y79 cells were tested by cell counting kit-8 (CCK-8) kit (Sigma, USA). In brief, the transfected HXO-RB44 and Y79 cells (1 × 104 cells/well) were planted into 96-well plates. At different time points of 24, 48, 72 and 96 h, 10 μL CCK-8 was added into each well. Subsequently, the cells were then cultured at room temperature for 1 h. Finally, the absorbance was measured at 450 nm by a microplate reader (Bio-Rad, USA).
Colony formation assay
The transfected HXO-RB44 and Y79 cells were respectively seeded into 6-well plates at a density of 300 cells per well and cultivated for 14 d. After fixed in 4% cold formaldehyde for 30 min, the colonies were stained with 0.1% crystal violet (Beyotime, China) for 20 min. Finally, the colonies was photographed and counted under an optical microscope (Olympus, Tokyo, Japan).
Cell cycle analyses
The transfected HXO-RB44 and Y79 cells were harvested, washed with PBS, fixed in pre-cold 70% (v/v) ethanol for 2 h at -20℃. Subsequently, the cells were stained with MuseTM Cell Cycle Reagent (Merck Millipore, USA) in dark for 30 min. Finnally, the cells were observed and analyzed by MUSETM flow cytometry (Merck Millipore, USA).
Apoptosis assay
The apoptosis of HXO-RB44 and Y79 cells was determined by Annexin V-FITC apoptosis detection kit (Invitrogen, USA). In brief, the transfected HXO-RB44 and Y79 cells were collected and resuspended in Binding buffer. Then, the transfected HXO-RB44 and Y79 cells were stained by using Annexin V-FITC and propidium iodide (PI) for 15 min in a dark room. Subsequently, the apoptotic cells were observed using MUSETM flow cytometer.
Real-Time fluorogenic PCR assays
Total RNA from RB tissue, adjacent normal retina tissue, HXO-RB44 cells, Y79 cells and ACBRI-181 cells was extracted using TRIZOL (Invitrogen, USA). Then, cDNA was synthesized from total RNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo, USA). Subsequently, the qRT-PCR was performed using SYBR green qPCR Master Mix (Thermo Scientific, USA) according to the manufacturer's protocol. Primers used in this study were as follows: lncRNA-UCA1 F: 5′-CCACACCCAAAACAAAAAATCT-3′, R: 5′-TCCCAAGCCCTCTAACAACAA-3′; GAPDH F: 5′-TGTTCGTCATGGGTGTGAAC-3′, R: 5′-ATGGCATGGACTGTGGTCAT-3′.
Western blot analysis
The total proteins were extracted by RIPA buffer (Beyotime, Jiangsu, China). 50 μg of protein samples were separated by 10% SDS-PAGE and then transferred onto nitrocellulose membrane. Subsequently, the membranes were blocked with 5% milk in TBST for 2 h, followed by overnight incubations at 4℃ with the primary antibody (PI3K, 1:1000; Akt, 1:1000; PCNA, 1:1000; GAPDH, 1:1000, Sino Biologial, USA. p-PI3K, 1:500; p-Akt, 1:500; S6K, 1:1000, Abcam, USA. survivin, 1:1000; CDK2, 1:1000; Caspase-3, 1:1000; p16, 1:1000; p21, 1:1000, Cell Signal, USA). Subsequently, the membranes were incubated with the peroxidase-labeled secondary antibody (anti-rabbit IgG, 1:5000, I5381MSDS, Sigma, USA) for 1 h. Finnally, the protein bands were detected by an enhanced chemiluminescence (ECL) kit (Thermo Fisher, USA).
Statistical analysis
All statistical analyses were performed using SPSS 22.0 Statistical Software (Chicago, IL). The results were presented as the mean ± SD. Statistical significance was tested using the one-way ANOVA or Student’s t-test. P < 0.05 was considered to be statistically significant.