30 patients with prostate cancer and 30 healthy volunteers were included in our study. We get human prostate cancer tissues and its adjacent normal tissues from 30 PC patients (age range: 39-68 years old; T1-T2: 17; T3-T4: 13), and obtained peripheral blood from 30 healthy subjects (all males; age range: 34-67 years old) and 30 PC patients respectively. We collected all the samples and then used liquid nitrogen to store them immediately. Patients with a history or current diagnosis of tumors, hypertension, diabetes, urinary tract diseases such, or related infectious or immune diseases were excluded. All patients were informed and offered the consents. Ethics Committee of Soochow University Affiliated Wuxi Ninth Hospital approved our study.
PC cell lines Du145 (ATCC; ATCC® HTB-81) and PC-3 (ATCC; ATCC® CRL-1435), prostate epithelial cells RWPE-2 (ATCC; ATCC® CRL-11610™) (from healty human) and HEK-293T cells were purchased from Procell Life Science&Technology Co., Ltd (Wuhan, Hubei, China). All cells were cultured in Dulbeccos Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA, USA) which containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) at 37 °C with 5% CO2.
Real time-quantitative polymerase chain reaction (qRT-PCR) (25)
Trizol reagent (CWIO, Beijing, China) was used to extract total RNA following the manufacturer’s protocol, and Nanodrop ND-2000 spectrophotometer (Thermo Scientific™, USA) was used to get the RNA quantity. Then we used miDETECT A Track™ qRT-PCR Kit (Guangzhou RiboBio Co., China) and RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific™, USA) to reverse-transcribe 2 μg total RNA into cDNA to analyze miRNA, lncRNA and mRNA expression respectively. Bio-Rad CFX Connect™ Real-Time PCR Detection System (Bio-Rad, USA) was used to perform qPCR with 2x SYBR Green qPCR Master Mix (Bimake, China), and U6 and GAPDH were used as the normalization. Primer sequences were listed as following:
U6 forward, 5’-GCTTCGGCAGCACATATACTAAAAT-3’;
GAPDH forward, 5’-CTTTGGTATCGTGGAAGGACTC-3’;
CAR10 forward, 5’-TCAGTGCTGCTCCTGAGAGA-3’;
miR-203 forward, 5’-ACACTCCAGCTGGGGTGAAATGTTTA-3’;
reverse, 5ʹ-TGGTGTCGTGGAGTCG-3ʹ. The 2–ΔΔCq method (26) was applied to calculate the gene expression.
Dual-luciferase reporter assay (27)
We inoculated the 293T cells into 24 well plates and cultured overnight, then transfected wild-type/mutant-type CAR10 vector and miR-203 mimic/mimic control respectively into 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 48 h, we followed the instructions and used Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) to measure the firefly (wavelength: 570 nm) and Renilla luciferase (wavelength: 480 nm) activities. Renilla luciferase was used as the internal reference.
CAR10-siRNA, control-siRNA, miR-203 inhibitor, inhibitor control, CAR10-siRNA+inhibitor control, or CAR10-siRNA+ miR-203 inhibitor were obtained from Genechem Co., Ltd, (Shanghai, China), and were transfected into Du145 and PC-3 cells respectively. We used six-well plates to culture cells then used lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) to perform transfection for 48 h.
Cell viability assay (28)
We used 96-well plates to culture cells (5x104 cells per well) and then used MTT assay to measure cell viability following the manufacturer’s instruction. Briefly, MTT (Beyotime) solution was added into each well and incubated for 4 h at 37 °C, then the medium was removed and 150 μl DMSO was added. We used multifunctional micro-plate reader (POLARstar OPTIMA; BMG, Offenburg, Germany) to measure the optical density (OD) at 570 nm.
Flow cytometer analysis (29)
We collected cells (1x106) and washed with PBS and then used 70% ethanol to fix them at 4℃ overnight. The percentages of cells that underwent apoptosis were determined by two color analysis. viable cells annexin V negative and PI negative, early apoptotic cells annexin V positive and PI negative, necrotic cells annexin V negative and PI positive, and late apoptotic cells annexin V positive and PI positive. Then we used Annexin V-FITC (FITC, R&D Systems Inc., Minneapolis, USA) and propidium iodide (PI, R&D Systems Inc., USA) to measure the cell apoptosis according to the manufacturer’s protocol. At last, the rate of apoptosis was detected using FACSCalibur flow cytometer (BD Technologies) and analyzed using FlowJo 7.6.1 software (FlowJo LLC).
Western blot assay (30)
Protease inhibitor (Beyotime) and RIPA lysis buffer (Beyotime) were used to extract the protein, and Pierce™ BCA Protein Assay Kit (Thermo Scientific™, USA) was used to quantify the protein concentration. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the equal amounts of protein. Then proteins were transferred to PVDF membranes (Millipore). Then the membranes were incubated for 1 h with blocking solution containing 5% nonfat dry milk and then were incubated at 4 °C overnight with primary antibodies: pro-Caspase3 (Cat no. ab32499; 1:1000; Abcam), cleaved-Caspase3 (Cat no. 9664; 1:1000; CST), GAPDH (Cat no. 5174; 1:1000; CST). After washing for three times using TBST, the membranes were incubated at 37 ℃ for 1 h with horseradish peroxidase-labeled secondary antibody (Cat no. 7074; 1:2000; CST). At last, ECL detection reagent (BioRad, USA) and densitometry (BioRad ChemiDoc XRS system) were used to visualize and quantify the signal respectively.
We used GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) to do the statistics, and used mean ± standard deviation (SD) to describe the variables. Student’s t-test or ANOVA analysis followed by Tukeys post-test was performed to get P values, and P < 0.05 represent statistical differences. We repeated each experiment for three times.