2.1 Subjects

The 217 healthy volunteers and 230 PCa patients included in this study were all from the Affiliated Wuxi People’s Hospital of Nanjing Medical University. Among the PCa patients, 114 volunteers underwent surgical castration and 116 underwent drug castration. They were followed up 1 year after treatment to determine whether they developed castration-resistant prostate cancer (CRPC). Before the study started, we had received the informed consent signed by the volunteers and obtained the approval of the hospital ethics committee.

Information and blood samples from all volunteers, as well as tumor tissue and para-carcinoma tissue (normal tissue adjacent to tumor tissue) from 114 PCa patients who underwent surgery, were well preserved.

2.2 Cell culture

RWPE-1 cells (human normal prostate epithelial cells) were purchased from SUNNCELL company and were cultured using the special medium (SNLM-569) from the same company. LNCaP and PC3 cells (PCa cells) purchased from ATCC were grown in RPMI 1640 medium with 10% FBS and 1% Pen/Strep. All cell lines were cultured in an incubator with 5% CO2 at 37 °C.

2.3 RT-qPCR

The mirVana™ miRNA separation Kit (ThermoFisher, America) was used to extract total RNA. MiRNA was reversely transcribed into cDNA using the microRNA reverse transcripti on Kit from the same company, and lncRNA and mRNA were reversely transcribed using the cDNA First Chain Synthesis Kit (Beyotime, China) for RT-qPCR. GAPDH was used as a housekeeping gene of LINC01518, CNKSR2, Ki-67, Bcl-2, and Bax. Hsa-miR-320a level was normalized by U6. Their expression levels were calculated by the formula 2−ΔΔCt. The sequences of primers were listed in Supplemental Table 1.

2.4 Prognostic assay

Receiver Operating Characteristic (ROC) curve and logistic regression analysis were used to evaluate the prognostic value of LINC01518 in PCa. The SPSS software was used to construct ROC curve of LINC01518 in predicting CRPC and to perform univariate and multivariate logistic regression analyses.

2.5 Cell transfection

The well-grown PCa cells were pre-inoculated in a 6-well plate (the density was about 25%). Transfection was performed the next day when the cell density was about 30–50%. The small interfering RNA of LINC01518 was synthesized by RIBOBIO Biological Company. The hsa-miR-320a mimics and inhibitor were purchased from MedChemExpress company. 120 µL OPTI-MEM medium and 5 µL RNA solution (or negative control) were added into the sterile 1.5 mL EP tube successively, and then 6 µL Lipofectamine 2000 (Beyotime) was added and gently mixed, sitting for 15 min. The old medium was replaced with serum-free medium (1869 µL/well). The transfection mixture was added to the 6-well plate to culture away from light for 6 h. After transfection, they were replaced with fresh serum-containing medium and continued to culture for 24–48 h for follow-up experiments.

2.6 Cell viability assay

PCa cells were inoculated into a 96-well plate with a density of 4 × 104 cells/mL after transfection. After 24 h, the original culture medium was discarded, and 100 µL culture medium containing 10% CCK-8 reagent (Solarbio, China) was added to each well and incubated at 37 °C for 2–4 h. After incubation, the light absorption value at 450 nm was measured by an enzyme-labeled instrument, and the cell viability was calculated according to the calculation formula in the CCK-8 reagent specification.

2.7 Migration and invasion assay

An uncoated or Matrigel-coated dual-chamber system (pore size 8 μm, Corning) was used for migration or invasion tests. 100 µL of FBS-free cell suspension (1 × 106 cells/mL) was added to the upper cavity, and 500 µL of medium containing FBS was added to the lower cavity. After 24 h, the migrated and invaded cells were fixed with 10% formaldehyde, stained with 5% crystal violet, and observed under a microscope, to count the number of osmotic cells from five selected fields at random.

2.8 Western blotting

Total protein was extracted using RIPA lysate (Solarbio), whose concentration was detected by a BCA kit (Solarbio). SDS-PAGE was performed with 50 µg protein per well. After the isolated proteins were transferred to PVDF membranes, they were incubated in 5% skim milk at room temperature for 2 h. The PVDF membrane was then incubated in primary antibody solution at 4 °C overnight. Next day, the PVDF membrane was incubated in the secondary antibody solution for 1.5 h, and the protein signal was detected by the ECL chemiluminescence detection kit (Absin, China).

2.9 Detection of apoptosis rate

The apoptosis rate was detected by an Annexin V-FITC/PI apoptosis detection kit (Absin). The cells were collected in an EP tube and cleaned with pre-cooled PBS buffer. After the supernatant was removed, 300 µL of 1× Binding Buffer and 5 µL of Annexin V-FITC were added and incubated for 15 min at room temperature away from light. Then 5 µL of PI solution was added to detect apoptotic cells using the flow cytometry.

2.10 Bioinformatics analysis

The GSE230278, GSE179321, and GSE115414 datasets were derived from NCBI-GEO Database (https://www.ncbi.nlm.nih.gov/geo/). The LncRNASNP2 Database (https://guolab.wchscu.cn/lncRNASNP#!/) was used to predict the downstream miRNAs of LINC01518. TargetScan-Human 8.0 Database (https://www.targetscan.org/vert_80/) and starBase Database (https://rnasysu.com/encori/index.php) were used to predict the downstream genes of hsa-miR-320a.

2.11 Double-luciferase reporter gene assay

Wild-type and mutant LINC01518/CNKSR2 gene fragments were cloned into double luciferase vectors to construct WT- and MUT-LINC01518/CNKSR2. WT- or MUT-LINC01518/CNKSR2 were co-transfected with miRNA negative control (mim-NC) or hsa-miR-320a mimics (mim-320a). The luciferase activity was then analyzed using the Dual-Luciferase Reporting System (Beyotime).

2.12 Statistical analysis

The significance of the association between various clinical factors and LINC01518 levels was analyzed by χ2 test using SSPS software. The difference between the two groups was analyzed by Student’s t-test, and the difference between multiple groups was analyzed by one-way ANOVA, using GraphPad software. All results were expressed as mean ± standard deviation (SD), and p < 0.05 were considered statistically significant.