Introduction
Prostate cancer is the most frequent malignancy among men in most developed countries, and it was estimated to contribute to 28% of newly diagnosed cancers and 11% of cancer-related deaths in 2011 [
1]. The progression-free survival rates are so short due to limited treatment strategies, which include surgery, radiation therapy and new therapeutic agents [
2,
3]. Although the etiology of prostate cancer is still unknown, growing evidence has indicated that multiple changes in specific genes in particular tumors are responsible for the development and progression of PCa [
4-
7]. There is an urgent need for new, more effective gene therapy programs which can be used in clinical applications.
MicroRNAs (miRNAs) are endogenous small non-coding RNAs of 18–25 nucleotides that act as posttranscriptional regulators of gene expression in diverse biological processes through imperfect base pairing with the 3′-UTR of target mRNAs, inhibiting target gene expression [
8,
9]. More interestingly, the roles of miRNA have proven to be indispensable in affecting cancer biology, including proliferation, autophagy, apoptosis, and invasiveness [
10-
14]. Accumulating data have suggested that miRNAs are involved in the tumorigenesis and progression of prostate cancer and act as a tumor suppressors or oncogenes [
15-
20]. MiR-203, a putative tumor suppressor gene, has been shown to inhibit cell proliferation and invasion and modulate the chemotherapy response in a variety of tumor cells, including lung cancer cells, glioma cells, and breast cancer cells [
21-
24]. At first, miR-203 has been identified as a skin-specific microRNA, and altering expression of miR-203 in vivo results in promoting epidermal differentiation by restricting proliferative potential and inducing cell-cycle exit through targetting p63, an essential regulator of stem cell maintenance in epithelial stratified tissues [
25]. It has been reported that in breast cancer miR203 targets SOCS3 (suppressor of cytokine signaling 3), a negative regulator of fetal liver hematopoiesis and placental development, and ABL1 (Abelson murine leukemia viral oncogene homolog 1), which implicates in processes of cell differentiation, cell division, cell adhesion, and stress response [
26,
27]. More importantly, miR-203 expression was also observed to be downregulated in prostate cancer tissues, and the over-expression of miR-203 significantly suppresses the growth and invasion of PCa cells [
28-
30]. Therefore, further exploring the function of miR-203 could broaden the strategies for prostate cancer treatment.
According to the mRNA sequence, Rap1A (Ras-related protein Rap-1A), member of RAS oncogene family, is a predicted target of miR-133a, which shares approximately 50% amino acid identity with the classical RAS proteins and has numerous structural features in common [
31]. Researches in leukocytes first demonstrated that Rap1 can enhance cell adhesion and migration and activate survival pathways [
32,
33]. RAP1 has been indicated to activate the MAPK/ERK pathway, which can contribute cell migration and inhibit cell differentiation [
34,
35]. In lung cancer, knocking down Rap1A can sensitize cancer cells to chemotherapy [
36]. It is also reported that activation of rap1promotes metastasis in prostate cancer and pancreatic cancer [
37].
The purpose of the present study was to verify the expression of miR-203 and investigate the molecular mechanisms through which it inhibits tumor growth and metastasis. Our data proved that Rap1A is a direct target of miR-203 and over-expression of Rap1A partly rescue the effect of miR-203 on cell proliferation, adhesion and invasion. All these indicate that miR-203 functions as tumor suppresser through inhibiting Rap1A in PCa and miR-203 may be used in clinical applications for cancer.
Methods
Cell lines and cultures
Human prostate cancer cell lines (22Rv1, LNCaP, PC3, DU 145), human HEK293T cells and nonmalignant epithelial prostate cell lines RWPE-1 were purchased from the American Tissue Culture Collection (ATCC) and were cultured in growth medium and under conditions recommended by the supplier.
Clinical samples
Paired tumorous and adjacent non-tumorous prostate tissues from 53 prostate cancer patients were obtained from the Tongji Hospital Subordinated to Tongji University with prior approval by the Ethics Committee of Tongji University and conducted according to the Declaration of Helsinki. The clinical diagnosis was made by a pathologist and confirmed by hematoxylin and eosin (H&E) staining. Surgical specimens used in this study were radical prostatectomy specimens and selected from the frozen prostate tumor bank if paired frozen blocks enriched for histologically normal and tumor areas were available.
RNA isolation and quantitative real-time PCR (qRT-PCR)
RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from prostate tissues and cells following the manufacturer’s protocol. The quantification of isolated RNA was assessed by a NanoDrop 2000 spectrophotometer (Nanodrop Technology, Inc., Wilmington, DE). Small RNA was converted to complimentary DNA using poly-A polymerase-based PrimeScript™ miRNA qPCR Starter Kit (TaKaRa). Approximately 500 ng total RNA was converted to cDNA using a PrimeScript™ One Step RT-PCR Kit (TaKaRa). Real-time qPCR was performed on the ABI Prism 7900HT (Applied Biosystems, Life Technologies, CA) using SYBR Green (TaKaRa) according to the manufacturer’s instructions. Small nuclear RNA U6 was used as an internal control for miR-203 and GAPDH was used to normalize Rap1A. Primers used for quantification were as follows: (Rap1A forward primer, 5′-GAAGAACGGCCAAGGTTTTGC-3′; Rap1A reverse primers, 5′-CCGTGTCCTTAACCCGTAAAATC-3′; GAPDH forward primer, 5′-GAAGGTGAAGGTCGGAGTC-3′; GAPDH reverse primers, 5′-GAAGATGGTGATGGGATTTC-3′). Each sample was run in three replicates with a dissociation curve analysis.
Plasmid constructs
To construct the has-miR-203 expression vector, the sequence containing the miR-203 pre-miRNA was amplified by PCR from human genomic DNA using the forward primer 5′-AAATCTAGACCAGGCGAGGGCGTCTAA-3′ and the reverse primer 5′-AAAAGAATTCAGCGGTTCCCACAGCACA-3′. The final PCR product was cloned into the XbaI/EcoRI sites of the pCDH-CMV-EF1-copGFP vector (SBI) according to the manufacturer’s instructions and named pCDH-miR-203.
A 555-bp fragment of the coding sequence of human Rap1A was amplified by RT-PCR using specific primers sense 5′-AAAGGATCCTATGCGTGAGTACAAGCTAGTGGT-3′ and anti-sense 5′-AAAAGAATTCCTAGAGCAGCAGACATGATTTCT-3′ and cloned into pCDNA3.1 (+) vector (Invitrogen) and named it pCDNA- Rap1A.
To construct the firefly luciferase reporter plasmids, the full-length 3′-UTR of Rap1A was subcloned into the pMIR-REPORT™ Luciferase (Lifetechnologies) to generate RAP1A-3′-UTR-WT. A mutant construct of Rap1A 3′-UTR, named RAP1A-3′-UTR-MUT, which carried a substitution of four nucleotides within the predicted miR-203 binding sites of Rap1A 3′-UTR, was carried out using a Phusion® site-directed mutagenesis kit (New England Biolabs). The primers for Rap1A 3′-UTR were 5′-AAAAAGCTTGCCCATAGTCAGCAGCAGCT −3′ and the reverse primer was 5′-AAAACGGCTTACAAGCTGTGGAAGATCTGAAATA −3′.
To knockdown Rap1A in the PC-3 and DU145 cell lines, oligonucleotides targeting Rap1A were designed and annealed to clone into the pLKO.1-TRC cloning vector at the AgeI/EcoRI sites with the forward primer 5′-CCGGCCCAACGATAGAAGATTCCTACTCGAGTAGGAATCTTCTATCGTTGGGTTTTTG-3′ and the reverse primer 5′-AATTCAAAAACCCAACGATAGAAGATTCCTACTCGAGTAGGAATCTTCTATCGTTGGG-3′. The scramble vector (SCR) was used as negative control. All sequences of plasmids were confirmed by DNA sequencing.
Generation of stable cell lines
The lentiviruses were produced by co-transfecting HEK293T according to the manufacturer’s instructions. For over-expression studies, the lentiviruses expressing miR-203 and control viruses expressing GFP were used to infect PC-3 and DU145 cell lines in the presence of 4 g/ml of polybrene (Sigma). The efficacy of miR-203 over-expression was assessed by qRT-PCR. For Rap1A silencing, PC-3 and DU 145 cell lines were infected as described above for miR-203. Puromycin (4 μg/ml) (Sigma-Aldrich, St. Louis, MO) was added 2 days after infection for the selection of infected cells. Western blots were performed to detect the efficiency of Rap1A in the selected cells.
Luciferase assays and transient transfections
HEK 293 T cells were co-transfected with 1 μg of RAP1A-3′-UTR-WT or RAP1A-3′-UTR-MUT plasmid and 50 ng of pRL-TK Renilla luciferase expression plasmid with an empty vector control or pCDH-miR-203 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Approximately 24 hours after transfection, cells were lysed, and luciferase activity was determined on a scintillation counter with the Dual-Luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. PC-3 and DU 145 cells overexpressed miR-203 were transfected with Anti-miRNA inhibitors Negative Control (anti-NC) and anti-miR-203 inhibitor (anti-miR-203) (Applied Biosystems) at a final concentration of 50 nM with the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Each reporter plasmid was transfected at least three times and each sample was assayed in triplicate.
Cell proliferation assay
Approximately 4 × 103 cells per well in triplicate were seeded in 96-well plates. Cell proliferation assays were assessed at 24, 48 and 72 hours using Cell Counting Kit-8 (CKK-8) (Dojindo, Rockville, MD). The relative number of cells was determined by measuring the optical density of each sample at 450 nm in a microplate reader (Beckman Coulter DTX880 Multimode Detector). All experiments were performed three times, and the average results were calculated.
Cell adhesion and invasion assay
Approximately 1 × 105 cells were harvested and resuspended in culture medium and then transferred to a 24-well plate that was coated with matrix proteins (40 μg/ml type I collagen or 2 μg/cm2 fibronectin in PBS) at room temperature for 1 h and washed twice with phosphate-buffered saline (PBS) to remove the non-adherent cells. The attached cells were measured by CCK-8 in a microplate reader at 450 nm. The adhesion of cells was related to the control set at 100%.
The cells (2 × 105/well) were suspended in 100 μl of serum-free medium and placed into the upper chamber of the insert with an 8-μm micro-porous membrane (Costar, Corning Inc., MA) coated with Matrigel (BD Bioscience, Bedford, MA). Medium containing 20% fetal bovine serum was added into the lower chamber. Following 24 hours of incubation at 37°C, the cells remaining on the upper surface of the membrane were removed with a cotton swab, and the migrated cells on the lower surface were fixed with methanol and stained with 1% crystal violet for 15 minutes. The stained cells were counted in five randomly selected fields per membrane under an inverted Olympus phase-contrast microscope (100× magnification). Each experiment was performed in triplicate wells and repeated three times.
Western blot analysis
Total cellular and tissue proteins were lysed in RIPA buffer in the presence of protease inhibitors (Roche Applied Science, Mannheim, Germany). Protein concentration was quantified using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Equivalent protein (50 ug) from each sample was loaded, and the lysates were separated by SDS–PAGE electrophoresis and transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked in 5% milk in Tris-buffered saline (TBST) containing 0.5% Tween-20 for 1 hour and incubated with primary antibody (RAP1A, Abcam; RAC1, p-MEK1, p-PAK1, GAPDH, Cell Signaling Technology; MEK1, PAK1, OriGene Technologies) overnight at 4°C. After 3 washes (10 min) in TBST, membranes were detected using horseradish peroxidase-linked goat anti-mouse or goat anti-rabbit IgG antibodies and visualized with the SuperSignal chemiluminescent system (Thermo Scientific, Rockford, IL). The intensity of the bands was analyzed using Bandscan software. All the shown images are from a single experiment that is representative of at least three separate experiments.
Tumor Xenografts in vivo
Mice were maintained in the animal facility and all experimental procedures using animals were approved by the Institutional Animal Care and Use Committee of Tongji University. DU 145 cells infected with vector or miR-203 at 5 × 106 cells/150 μl were injected subcutaneously into the left flanks of anesthetized 6-week-old NOD-SCID mice. Mice were sacrificed 5 weeks later and tumor weight was determined in grams at the end of the experiment. Tumor samples were excised for western blotting.
Statistical analysis
All data were presented as the means ± S.D of three or more replications. For relative gene expression, paired Student’s t test was used to determine the statistical significance and two-tailed unpaired Student’s t tests and analysis of variance were used for statistical evaluation of the data. Pearson's correlation analysis was performed to examine the correlation between miR-203 and Rap1A mRNA levels of human PCa tissues. Data analysis was performed using GraphPad Prism 4.0 (Graph Pad Software, La Jolla, CA) and two-tailed p values < 0.05 were considered to be significant.
Discussion
MiRNAs play important roles in a broad range of biological processes including cellular differentiation, proliferation, apoptosis, and tumor development. In the present study, our results provided the first evidence that miR-203 supresses cell proliferation, adhesion and invasion through targeting Rap1A in PCa.
First we verified that the expression of miR-203 was significantly decreased in cancerous tissues of PCa patients compared with non-cancerous prostate specimens. In addition, we found that miR-203 was reduced in PCa cell lines with high metastatic potential compared with the noncancerous prostate epithelial RWPE-1 cells. To further investigate the mechanisms of miR-203 downregulation in PCa, we over-expressed miR-203 in PCa cells and assessed various aspects of PCa cell biology. Introducing miR-203 into PC-3 and DU 145 cells, which express relatively low levels of endogenous miR-203, led to a significant inhibition of cell proliferation, adhesion and invasion. Data indicates that miR-203 functions as tumor suppressor in PCa and is down-regulated in advanced PCa.
Cell adhesion and invasion both play essential roles in tumor growth and metastasis. Then, we focused on the targets of miR-203 which may modulate cellular adhesion and invasion in the present study. Based upon bioinformatics analysis, Rap1A was predicted as a putative miR-203 target and the putative target sites for miR-203 presents within Rap1A 3′-UTR. Rap1 is a member of the Ras family of small GTPases and was activated in response to a number of extracellular stimuli [
31]. Many studies have shown that Rap1A was upregulated and involved in cell-cell and cell-extracellular matrix adhesion in many cancers [
32-
34]. By firefly luciferase activity assay, miR-203 could reduce luciferase activity in the Rap1A wildtype but had no effect on the mutant construct. Meanwhile, the over-expression of miR-203 could lead to a decrease of endogenous Rap1A expression in PCa cells at both the transcriptional and translational levels. Furthermore, miR-203 expression was inversely correlated with Rap1A mRNA expression in PCa tissue specimens. All these results proved Rap1A is a novel direct target of miR-203.
It is complicated to understand the biogenesis of special miRNAs because different direct targets of certain miRNA may play different roles in different organs and different developmental periods. To further validate whether miR-203 plays a tumor-suppressive role by inhibiting Rap1A expression, knockdown Rap1A by shRNA-mediated Rap1A downregulation was confirmed at protein levels. Functional analyses were performed and found Rap1A silencing could phenocopy the effects of miR-203 on phenotypes of PCa cells. Recent reports have shown that the activation of Rap1A was increased in PCa and Rap1A up-regulated the expression of integrins to promote CPa migration and invasion in vitro and in vivo [
37]. Here we focused on the roles of Rap1A in cell adhesion and invasion. We have shown that miR-203 over-expression reduced the activation of Rac1/PAK1 pathways. Consistent with this, we also observed inhibition of Rap1A resulted in inactivation of Rac1/PAK1 pathways. Importantly, the significant effects induced by miR-203 over-expression on cell adhesion and invasion were partially reversed by over-expression of Rap1A. All these proved that Rap1A is a functional target of miR-203 and this evidence suggested that miR-203 exerts its tumor-suppressive effects through downregulation of Rap1A in PCa cells. Xenograft assay proved such regulation in vivo too.
Briefly, we found that Rap1A is a novel functional target of miR-203 and both over-expression of miR-203 and knockdown of Rap1A have similar effects in PCa, which is suppressing cell proliferation, adhesion and invasion through inactivation of the Rac1/PAK1 signaling pathway. All data elucidate a new mechanism that miR203 suppresses tumor growth through inhibiting Rap1A. Nevertheless, additional direct target genes mediated by miR-203 remain to be further identified in the future.
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Competing interests
All authors declare that they have no competing interests.
Authors’ contributions
JX and CDB initiated the study, carried out the study, performed the statistical analysis, and drafted the manuscript. HW and SSH carried out the study and statistical analysis. DLW participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.