Cell culture
RWPE-1 cells were grown in Keratinocyte Serum Free Medium (Gibco, Invitrogen, USA) supplemented with bovine pituitary extract and human recombinant epidermal growth factor to make the complete medium. C4-2 cells were grown in Dulbecco’s Modified Eagle’ medium (DMEM): F12 medium (1:1) (Invitrogen, USA), LNCaP and 22Rv1cells were maintained in RPMI1640 (Invitrogen, USA) while DU 145 cells were maintained in Eagle’s Minimum Essential Medium (Thermo Fisher Scientific, USA), and PC-3 cells were grown in F-12K medium (Thermo Fisher Scientific, USA). Except for RWPE-1 cells, all other mediums were supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin (Gibco- Thermo Fisher Scientific, USA). All the cells were cultured under standard laboratory conditions in an incubator at 37 °C with 5% CO2 supply.
Collection of clinical samples
60 pairs of tissue samples from PRAD patients were collected at Taikang Tongji (Wuhan) Hospital. Samples were collected after surgical removal of PRAD and adjacent tissue samples which were immediately stored at -80°C until further use. All the clinical procedures were in accordance with the declaration of Helsinki and written informed consents were collected from all the participants of the study. All the protocols were reviewed and approved by the committee for human experimentations, Taikang Tongji (Wuhan) Hospital, China.
Cell viability assay
Cell viability was determined by the cell counting kit 8 assay (CCK-8). Briefly, 6 × 103 cells were seeded into 96 well cell culture plates and cultured for an overnight. Different treatments were applied and cells were incubated for 24, 48, and 72 h. At the end of incubation period, 10 µL of CCK-8 (Dojindo, Japan) was added to each well and further for 1h at 37 °C. Finally, the absorbance was measured at 450 nm wavelength using Elx800 absorbance reader (BioTek Instruments).
Colony formation assay
This assay was conducted to investigate the proliferation capacity of the cells. For this purpose, PRAD cells transfected or not were seeded onto 24-well plates with 500 cells per well. Cells were cultured for 10 days under standard laboratory conditions. The, cell were fixed with 100% methanol (Sigma, USA) and colonies were visualized under laboratory microscope (Olympus, Japan)after staining with 0.1% crystal violet solution (Solarbio, China).
Western blot assay
Collected cells were lysed in ice cold RIPA buffer (Servicebio, China) with added protease inhibitor cocktail (Sigma, USA). Protein content of samples was determined by using BCA assay kit and proteins were separated by electrophoresis in 4%‐20% polyacrylamide gels (GenScript). Afterwards, the proteins were transferred to polyvinylidene fluoride membranes (Millipore, USA) followed by saturation in 5% skim milk at room temperature. Then, primary antibody (1:500) was added to the membranes and incubated for an overnight at 4°C. Then, the secondary antibodies (1:2000) and incubated at room temperature for 2 h. Protein blots were detected using Enhanced Chemiluminescence Kit (FDbio Science, China).
Flow cytometery assay
Cells were collected after 72 h of transfection or not and washed three times with PBS. Cells were then incubated in dark with FITC‐Annexin V and propidium iodide (Liankebio, China) for 15 min. Flow cytometery was performed using FACSCantoⅡ flow cytometer (BD, USA) and FlowJo 10.0 software was used to determine the percent of apoptotic cells.
Subcutaneous tumorigenesis assay in nude mice
BALB/c male nude mice weighing approximately 22-25 g were purchased form the Zhejiang Chinese Medical University (Hangzhou, China). Mice were acclimatized for at least a week under standard laboratory conditions. Afterwards, 2 × 106 PRAD cells suspended in 200 cells μL of PBS were subcutaneously injected into the left flank of the mice. Tumor xenograft diameter was measured after each week for a total of 4 weeks for determining the tumor volume using V = π/6 × length × width2 and tumor weight was also determined at the end of this period. All the experimental protocols were approved by the animal ethical committee of Taikang Tongji (Wuhan) Hospital and were in compliance with the ARRIVE guidelines.
Quantitative reverse transcription polymerase chain reaction (RT‑qPCR)
TRIzol reagent (Invitrogen, USA) was used for the extraction of total RNA from the samples and the RNA concentrations were determined by using nano-drop spectrophotometer. High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) was used to reverse transcribe RNA to cDNA. This was followed by PCR for 40 cycles of alternate temperatures of denaturation, annealing and extension. Gene expression was analyzed employing SYBR Green PCR master mix (Thermo Fisher Scientific, USA) using 2-ΔΔCt method. Product specificity was analyzed by melt-curve analysis and all the experiments were replicated at-least in triplicate.
Luciferase reporter gene assay
Approximately 5 × 104 cells were seeded onto a 96-well plate for 24 h followed by transfection/co-transfection for 48 h with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s guidelines. Luciferase assay kit (Promega, USA) was used to perform the luciferase activity according to the provided protocols. In brief, to a volume of 100 µL containing cells an equal volume of luciferase assay solution was added and incubated for 20 min at room temperature. Microplate reader (Synergy H4 Hybrid Reader, BioTek, Winooski, USA) was used to measure luciferase activities [17].
RNA pull-down assay
Previously described procedures were used for the RNA pull-down assay [18]. This assay was conducted according to the previously described procedures. Biotin-labeled bio- LINC01213 probe was provided by Sangon Biotech (Shanghai, China). Cells were collected and lysed. One part of the lysate was kept to be used for input control. The other part was incubated with magnetic Dynabeads M-280 Streptavidin beads (Invitrogen, Carlsbad, CA, USA) beads for an overnight at 4 °C. qRT-PCR assay was used to determine the miR-597-3p enrichment.
Statistical analysis
Statistical analysis of the results was conducted by the GraphPad prism software version 6. Kaplan–Meier analysis was used to compare the survival difference between patient groups. Student’s t test was used for two groups while One-way ANOVA was used for the statistical analysis of more than two groups. P values less than 0.05 were considered significant. The data represent the mean ± SD of at-least three independent experiments.