Clinical samples and cell culture
Clinical samples were obtained from 5 patients with BPH (benign prostatic hyperplasia), 12 prostate cancer patients with low Gleason score and 12 patients with high Gleason score came from the Second Hospital of Tianjin Medical University. All patients provided informed consent for the study, which got the approval of Research Ethics Committee of the Second Hospital of Tianjin Medical University. The human prostate cancer cell lines, LNCaP, 22Rv1, C4-2, DU145, PC-3, and human benign prostatic hyperplasia cell line BPH-1, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). 22Rv1 and DU145 cells were maintained in RPMI-1640 (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37 °C, 5% CO2. LNCaP-AI cells were established by LNCaP cells in RPMI medium supplemented with 10% charcoal-dextran-stripped FBS (CD-FBS) for more than 12 months at 37 °C, 5% CO2.
Protein extraction and western bolt assays
Western blot was performed with whole-cell protein lysates in RIPA lysis buffer (Thermo Fisher Scientific, Inc.) and lysed by centrifugation at 14,000 rpm for 30 min at 4 C.The polyacrylamide gel was prepared by Solarbio kit (P1200, Solarbio, China) Equal amounts of 30 µg proteins were fractionated by SDS–PAGE, then transfected to polyvinylidene difluoride membranes. After blocked in 5% Skimmed milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. Then secondary antibodies were incubated for 1h in room temperature. Use immobilon western chemilum hrp substrate (WBKLS0100, Millipore) to exposure and photography. All experiments were performed three repetitions.
Stable and transient transfection
For shRNA transfection, PC-3 and LNCaP-AI cells were transfected with lentiveiral shRNA purchased from GENECHEM (Shanghai, China) when the cells reaching 40-60% confluency. Plasmids were transiently transfected into prostate cancer cells using Lipofectamine2000 (Thermo Fisher Scientific, Inc.) For preparation, cells were grown in complete medium for at least 24 h, and washed 3 times by phosphate buffered saline (PBS; pH 7.4) before transfection. Stable expression was selected with puromycin after transfection. All experiments were performed three repetitions.
RNA extraction and Quantitative Real Time Polymerase Chain (qRT-PCR) Assay
To detect the expression levels of miR-326 and CKAP2L, qRT-PCR assay was performed. Total RNA was extracted from the cells with TRIzol (Invitrogen Life Technologies, USA) according to the manufacturer’s instructions. The reverse transcription kit used in the reverse transcription process was purchased from Thermo Fisher Scientific. Then cDNA amplification was conducted using the SYBR-Green PCR Master Mix (Roche) with Applied Biosystems 7900 Real Time PCR System (Thermo Scientific). The relative expression level was analyzed using 2−ΔΔCt method and normalized with U6 and GAPDH as endogenous control. All experiments were performed three repetitions.
CKAP2L IHC was performed with the standard streptavidin-biotin-peroxidase complex method by the immunohistochemistry kit (pv-6000, ZSGB-BIO, China) The nuclear accumulation of CKAP2L was assessed according to the ratio of prostate cancer cells with positive nuclear staining. The results were scored by the positive cell rate and staining intensity.
Cell proliferation assay
Transfected cells were digested, resuspended and then plated into 96-well plates at a density of 8 × 103 cells/well with six repeats. At 0, 24, 48, and 96 h, 10 μL of MTT regent (5 mg/mL) was added into per well and cells were incubated at 37°C for 2 h. Thereafter, the supernatant was removed and the precipitate was solubilized in 200 μL of dimethyl sulfoxide (DMSO). Finally, the absorbance value of each well was measured at a wavelength of 490 nm.
Colony formation assay
The colony formation assays were performed to test the colony formation ability of cells. 500–1000 cells were seeded per well in 60 mm plates and grown for two weeks. Then cells were fixed with methanol for 15 min and stained with 1% crystal violet-acetic acid solution for 20 min, colonies were visualized and quantitated by ImageJ. All experiments were performed three repetitions.
Migration assay: Cells on logarithmic growth phase were performed with serum starvation for 24h. On the next day, cell suspension with concentration of 4 × 104 cells/mL was prepared. 0.2 mL of suspension was added into the Transwell inserts, and 800 μL of pre-cooled 1640 containing 10% FBS/CD-FBS was placed out of the inserts. After 48 h of incubation in 5% CO2 at 37°C, unmigrated cells were wiped off with a cotton swab, meanwhile, cells migrated out of the inserts were fixed by methanol at 4°C for 30 min and stained in 0.1% crystal violet for 20 min. Images were captured with an inverted microscope, and three fields were randomly selected for cell count. All experiments were performed three repetitions.
Invasion assay: Roughly 2 × 104 cells were transfused into the upper chambers with Matrigel matrix (Corning, NY, USA) precoated. 800 μL of pre-cooled 1640 containing 10% FBS/CD-FBS was placed in the lower chambers. Following steps were similar with migration assay as mentioned above. All experiments were performed three repetitions.
RNA-Binding Protein Immunoprecipitation Assay (RIP)
RIP assay was performed using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore 3480215, Billerica, USA) as described by the manufacturer protocol. Briefly, cells were lysed with complete RIP lysis buffer. 100 μl whole cell lysate from each group were incubated with RIP buffer containing magnetic beads conjugated to mouse anti-Ago2 antibody (ab186733, 1:50, Abcam, United Kingdom), or negative control normal rabbit IgG (Millipore, USA). Consequently, the immunoprecipitation was digest by incubated with proteinase K to purify the immunoprecipitated RNA. Purified RNA was reverse transcribed into cDNA and subjected to qPCR to examine the presence of CKAP2L mRNA. All experiments were performed three repetitions.
Primers and antibodies
All primer and antibody details are listed in supplementary Table 1.
Luciferase reporter assay
The putative has-miR-326 binding sites in 3′UTR of CKAP2L, as well as the mutant binding sites were sub-cloned into pmirGLO luciferase vector (Promega, Madison, WI, USA)to construct luciferase expression plasmids WT-CKAP2L and MUT-CKAP2L. Then co-transfected them into prostate cancer cells with miR-326 mimic or miR-326 mimic NC. Lipofectamine 2000 was used for transfection according to the instructions. After 48 h, a luminometer (Beckman Coulter LD400, CA, USA) was used to detect the luciferase activity. All experiments were performed three repetitions.
Cell Cycle Assessment
PC3 and LNCaP-AI cells were harvested, fixed with 70% ethanol, briefly washed with PBS/1% FBS, and then incubated with 10 mg/mL RNAse A and 50 mg/mL propidium iodide in PBS plus 1% Tween 20 for 30 min at 37 °C in the dark. Flow cytometric analysis was performed using FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The cell fractions in the respective cell cycle phases were calculated using Cell Quest Pro software (BD Biosciences, USA). All experiments were performed three repetitions.
To check the inhibition effect on tumor growth after CKAP2L depletion, CKAP2L was downregulated by shRNA-mediated knockdown in PC3 cells. Then the shCKAP2L transfected cells and their counterparts were subcutaneously injected into the Balb/c nude mice (6 to 8-week old). The tumor weights were measured after the tumor formation.
The RNA expression from GSE21034 was transformed into log2(count+1). The RNA expression from TCGA database was transformed into log2(FPKM+1). Associations between CKAP2L or miR-326 expression and clinicopathological parameters were achieved by nonparametric analysis. Kaplan–Meier curves were fitted to disease free survival data. Statistical difference of two groups was calculated by two-tailed Student’s t-test and correlation of expression was analyzed by Spearman correlation test. ANOVA was used for multiple groups , and the error bars mean ±SD. Differences were considered significant when P < 0.05. Analyses were performed using GraphPad Prism 8 software (Intuitive Software for Science) and R-3.6.0.