PCa tissues samples
A total of 12 prostate cancer tissues and their paired adjacent tissues were collected from patients undergoing the radical prostatectomy at the Third Affiliated Hospital of SooChow University from Jun 2019 to Dec 2019. Patients enrolled in this study received no preoperative radiotherapy and/or chemotherapy. Each tissue was snap-frozen immediately in liquid nitrogen after excision, and stored at -80ºC until use.
Bioinformatic analyses
Gene expression and clinicopathological features of patients with prostate cancer in PRAD-TCGA were downloaded from UCSC Xena Cancer browser (https://xenabrowser.net). Microarray datasets (GSE21036 and GSE64318) containing miR-183-5p expression profiles of prostate biopsy samples (52 cancer tissues and 52 adjacent tissues) were downloaded from Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/gds/). SPSS17.0 (Chicago, IL, USA) and GraphPad Prism 5.0 (San Diego, CA, USA) software was used for the statistical analysis. Moreover, TargetScan, miRDB, miRWalk and StarBase were used to predict miR-183-5p target genes.
Cell lines and transfection
Human PCa cell lines, including androgen-dependent LNCaP and androgen-independent PC-3, were obtained from the Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in F12K or RPMI-1640 medium (Gibco, Carlsbad, CA) containing 10% fetal bovine serum (BI, Israel) at 37ºC with 5% CO2.
MiR-183-5p mimics, mimics negative control (miR-NC), miR-183-5p inhibitor and inhibitor NC were purchased from GenePharma (GenePharma, Shanghai, China). Cells were seeded in 6-well plates and grown to a density of 40–60%, then transfection was performed using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions, respectively. And these cells were harvested for verification of transfection efficacy after 48h.
CCK-8 assay
Cells were seeded in a 96-well plate at a density of 1×104 cells/well, and culture medium was used as a blank control. CCK-8 was added to each well (six replicate wells per group) at the indicated time points (10 µl/well), and the absorbance at 450 nm was measured 2 h later to estimate viable cells using an automatic plate reader.
Cell migration and invasion assay
A transwell chamber with an 8-µm pore size polycarbonate membrane (Corning, NY) was used to evaluate cell migration. After 48h of transfection, the PCa cells were resuspended with basic medium and seeded into the upper chamber (2×104 for PC-3 cells and 5×104 for LNCaP cells), while culture medium containing 15% FBS was added to the lower chamber. After incubation for 48 h at 37°C, the medium was removed from the upper chamber, and non-migrated cells were scraped off with a cotton swab. The migrated cells on the other side of the membrane were fixed in 4% paraformaldehyde for 30 min, stained with crystal violet for 15 min, and counted under an inverted microscope at 200x magnification in at least three randomly selected fields.
In the invasion assay, the upper chamber was pre-coated with 10% Matrigel (BD Biosciences, San Jose, CA, United States) and the procedures of the cell invasion assay were identical to the cell migration assay.
Wound healing assay
Transfected cells were plated into 6-well plates and grown to 90% confluence. A 10 µl sterile pipette tip was used to create a scratch wound across cell monolayer, then detached cells and debris were removed by PBS, and the attached cells were incubated in the medium with 1% FBS. Images of the wound closure was captured at 0 h and 48 h using the microscope.
Luciferase reporter assay
To verify whether miR-183-5p directly binds to the 3’UTR of TET1, luciferase reporter assay was performed. MiR-183-5p mimics or mimics negative control (NC) were transfected into HEK293T cells together with TET1 (wild type (WT) or mutant (MUT)) using lipofectamine 2000 (Invitrogen), respectively. The relative luciferase activities were measured using a detection kit (Promega Corp, USA) after 48 h, according to the manufacturer’s instructions.
Western blot analysis
The cells were harvested and lysed using protein RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, USA). The protein concentration was determined by the BCA protein assay kit (Beyotime, Shanghai, China). Samples were mixed with SDS-PAGE loading buffer (Beyotime, Shanghai, China) and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After transferring onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA), the membranes were blocked with 5% skim milk in TBST at room temperature for 1 h, and then incubated with anti-TET1(MA5-16312, 1:1000 dilution, Thermo Fisher Scientific) or anti-β-actin (ARG65683, 1:1000 dilution, Arigo) antibodies for 2 h, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h. Protein bands were visualized by an ECL + Plus western blotting detection system (CW Biotech, Beijing, China) and quantified using a scanner with Quantity One software (Version 4.2.1, Bio-Rad Laboratories, Hercules, CA, USA).
Total RNA extraction and real-time PCR (qRT‐PCR)
Total RNA was extracted from PCa cell lines and clinical tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using the RevertAid™ first strand cDNA synthesis kit (Thermo Fisher Scientific, USA). The obtained cDNA was subjected to qRT-PCR by performing on a LightCycler480®II in a final volume of 25 µL. Optimum reaction conditions were obtained with 0.04 µL of 100 µM of each primer and probe, 2.5 µL of 10× PCR buffer, 2.5 µL of 25 mM MgCl2, 0.5 µL of 10 mM 4× dNTPs, 0.25 µL of 5 U/µL Taq DNA polymerase, and 2 µL template cDNA. Finally, 17.13 µL ddH2O was added to the reaction mixture. The mixture was preheated at 95ºC for 3 min to activate Taq polymerase, followed by 40 cycles at 95ºC for 5 s and 60ºC for 15 s. Samples were amplified simultaneously in triplicate in one assay run. Data were normalized to GAPDH or U6. Sequences of primers and probes are summarized in Table 1.
Table 1
Sequences of primers and probes for real-time RT-PCR
Gene | Primer / Probe | Sequence (5’ to 3’) |
miR-183-5p | Forward | CCTGTTCTGTGTATGGCACTGGT |
Reverse | TTCACTGACTGAGACTGTTCACAGTG |
Human TET1 | Forward | CCATCTGTTGTTGTGCCTCTG |
| Reverse | GCCATTTACTGGTTTGTTGTCA |
| Probe | FAM-AGGTTATAAAGGAAAACAAGAGGCCCC-BHQ-1 |
Human GAPDH | Forward | CAACTACATGGTTTACATGTTC |
Reverse | GCCAGTGGACTCCACGAC |
Probe | CY5-TTTGGTCGTATTGGGCGCCTG-BHQ-1 |
Statistically analysis
Data were presented as mean ± S.E.M., and GraphPad Prism (version 5.0) was used for data analyses.
The paired Student’s t-tests were performed to compare two related samples, while the unpaired Student’s t-tests were used to compare differences between different groups. A one-way ANOVA test was conducted to compare the intergroup difference more than two groups. Kaplan– Meier curves were introduced for survival analysis. P value less than 0.05 was considered as statistically significant.