Patients and tissue samples
Total 69 paired of tumor tissues and matched adjacent normal tissues were obtained from patients with PCa who underwent radical prostatectomy at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Hubei, China). Before the surgery, all patients were confirmed not to receive chemotherapy, radiotherapy or androgen deprivation therapies. All tissue specimens were immediately frozen in liquid nitrogen and kept at -80 °C until further analysis. The basic clinicopathological characteristics of all PCa patients, including age, Gleason score and TNM stage were summarized in Table 1. All enrolled patients underwent five-year follow-up period through the telephone surgery. The written informed consent was signed by all patients and the present study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Hubei, China).
Cell culture
Human PCa cell lines (PC-3, 22RV1, DU145 and LNCaP) and one normal prostate epithelial cell line RWPE-1 were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco) in a humidified incubator containing 5% CO2 at 37 °C.
Cell transfection
MiR-5195-3p mimics, scrambled miRNA (miR-NC), smaller interfering RNA against CCNL1 (si-CCNL1), negative control (si-NC), the overexpression plasmid of pcDNA3.1-CCNL1 and empty vector pcDNA3.1 were synthesized from Shanghai GenePharma Co., Ltd. For cell transfection, PC-3 and DU145 cells were seeded into six-well plates and transfected with the above oligonucleotides using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.
Quantitative real-time PCR
Total RNA was extracted using mirVana miRNA isolation kit (Life Technologies; Thermo Fisher Scientific, Inc.) for miRNA abundance and RNeasy mini kit (Qiagen, Valencia, CA, USA) for mRNA. The synthesis of complementary DNA was performed using miScript II RT kit (Applied Biosystems, CA) for miRNA and superscript VILO cDNA kit (Thermo Fisher Scientific, Inc.) for mRNA according to the manufacturer's instructions. Quantitative real time PCR was performed using miScript SYBR Green PCR kit (Qiagen) for miR-5195-3p or SYBR Green PCR kit (Applied Biosystems, CA) for CCNL1 mRNA levels with the specific primer sequences synthesized by Sangon Biotech (Shanghai). Each experiment was performed in triplicate, and relative abundance was normalized to U6 for miR-5195-3p or GADPH for CCNL1 mRNA by the 2–ΔΔCT method.
Cell proliferation assay
Transfected PCa cells in technical triplicates at a density of 3 × 104 cells per well in six-well plates and cultured for 24, 48 and 72 h, respectively. At each time point, cells in each well were incubated with 10 µl of CCK-8 solution (Sigma-Aldrich) for 2 h. Afterwards, the absorbance at a wavelength of 450 nm was measured in each well using a microplate reader.
Cell cycle analysis
Transfected PCa cells at a density 4 × 105 cells per well were seeded in six-well plates and incubated undisturbed for 48 h. Subsequently, cells were washed with PBS, fixed with cold 70% ethanol overnight, followed by incubation with 0.1 mg/ml propidium iodide (Sigma-Aldrich) for 30 min in the dark. Next, the cells were analyzed by a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with FlowJo software (Version 10.0.4; FlowJo LLC).
Luciferase reporter assay
According to the predicted putative binding sites for miR-5195-3p with the 3′‐UTR of CCNL1 by the online software program TargetScan 7.1 (http://www.targetscan.org), we performed luciferase reporter assay to validate the above prediction. In brief, the fragments of CCNL1 3′‐UTR containing either putative miR-5195-3p seed sequence or corresponding mutant (MUT) sites using QuickChange Site-Direct Mutagenesis Kit (Stratagene) were subcloned into psiCHECK‐2™ vector (Promega, USA) to obtain the reporter plasmids of CCNL1-wild-type (WT) and CCNL1-MUT. Then, PC-3 or DU145 cells were plated in 24-well plates and co-transfected with 1 μg reporter plasmid CCNL1-WT or CCNL1-MUT together with 30 nM miR-5195-3p mimics or miR-NC for 48 h. Relative luciferase activities were determined using a Dual‐Luciferase Reporter Assay System (Promega).
Tumor xenograft formation
Equivalent amounts of PC-3 cells (1.8 × 106) stably transfected with miR-5195-3p mimics or miR-NC were subcutaneously injected into the right flank of 4–5-week-old nude mice (Shanghai Laboratory Animal Research Center, Shanghai, China) with five mice in each group. Mice were monitored every five days and the tumor length/width was measured using calipers. Tumor volume was calculated using the modified ellipsoid formula: Volume = 1/2 (length × width2). All mice were sacrifice 30 days after injection. Then, the tumor weight was measured and tumor tissues were harvested for analysis of Cyclin L1, CDK4 and Cyclin D1 expression. All animal experiments were performed in accordance with the Huazhong University of Science and Technology Research Institute Animal Care Committee guidelines.
Western blot analysis
Total protein samples were extracted from cell lines or tumor tissues with RIPA lysis buffer (Thermo Fisher Scientific, Inc.) and protein concentration was analzyed using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Then, equal amount of protein sample was subjected to electrophoresis using sodium dodecyl sulfate polyacrylamide gels (SDS‐PAGE), which was subsequently transferred onto PVDF membranes. After blocked with 5% nonfat dried milk in TBST for 2 h, the membranes were incubated with primary antibodies against Cyclin L1, CDK4, Cyclin D1 and GAPDH overnight at 4 °C, followed by incubation with horseradish-peroxidase-linked secondary antibodies for 2 h at room temperature. Afterwards, the protein bands were visualized by a chemiluminescence detection kit (ECL, Millipore, USA).
Statistical analysis
The GraphPad Prism 6.0 software (National Institutes of Health, Bethesda, MD, USA) was used to perform all statistical analysis. The association between miR-5195-3p expression and PCa clinicopathologic characteristics was assessed by Chi-square test. The Kaplan‐Meier method was performed to generate survival curves. Univariate and multivariate Cox regression models were constructed to estimate the hazard ratios (HRs) of independent factors affecting the overall survival in PCa patients. The Spearman’s correlation coefficient was used to analyze the association between miR-5195-3p expression and CCNL1 expression in PCa tissues. All the quantitative data were expressed as mean ± SD of at least three experimental replicates. The differences among groups were analyzed using either the one-way ANOVA or the Student’s t-test. Statistical significance was defined as p-value less than 0.05.