miR-574-3p is Significantly Down-regulated in PCa Tissue Specimens

We evaluated expression levels of miR-574-3p in human PCa tissues (n=48) and adjacent non-cancerous tissues (n=48). The expression level of miR-574-3p was significantly lower in PCa compared with normal tissues (P<0.0001; Fig. 1C). To determine if the levels of miR-574-3p in tumor tissues correlates with clinic-pathological factors, we analyzed miR-574-3p expression levels in human tumor samples. Clinical demographics of the study cohort are summarized in Table 2. Correlation of 574-3p expression with clinicopathological variables such as pathological stage (pT) and Gleason score is shown in Fig. 1D. These results reveal that cases with low miR-574-3p expression increase from low grade, low pathological stage to high grade and high pathological stage. These results suggest that miR-574-3p is significantly down-regulated in PCa and may be a putative tumor suppressor in PCa.

Table 2

Prostate cancer patient information.

Characteristics			(%)
Age (years)
Median (range)	62 (47–81)
PSA (ng/ml)
Median (range)	7.0 (0.2–90)
Total number	48		(100.0)
Gleason Score
GS 6	23		(47.9)
GS 7	15		(31.3)
GS 8	6		(12.5)
GS 9	3		(6.2)
unknown	1		(2.1)
Pathological tumor stage
pT2a	8		(16.7)
pT2b	10		(20.8)
pT2c	13		(27.1)
pT3a	8		(16.7)
pT3b	1		(2.1)
unknown	8		(16.7)

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Abbreviations: PSA=prostate-specific antigen; GS=Gleason Score.

Effect of miR-574-3p Over-expression on Cell Proliferation, Migration, and Invasion in PCa Cell Lines in vitro and in vivo

To examine the functional roles of miR-574-3p, we performed gain-of-function studies using a pre-miR-574-3p miRNA precursor transfected into PC3 and DU145 cells. The expression of miR-574-3p was markedly up-regulated in Pre-miR miRNA precursor transfectants (Fig. 2A; PC3 316.8-fold, DU145 307.5-fold). Cell proliferation assay (MTS) and wound healing assay showed significant inhibition in miR-574-3p transfectants in both the PC3 and DU145 cells compared to the control transfectants (Fig. 2B and 2C). Invasion assay (Matrigel) also showed that the number of invading cells was significantly decreased in miR-574-3p transfectants compared with their counterparts (Fig. 2D). To confirm the effect of miR-574-3p on tumorigenicity in vivo, miR-574-3p and miR-control-transfected DU145 cells were subcutaneously injected into nude mice. We observed that miR-574-3p over-expression inhibited DU145 cell tumor formation in vivo (Fig. 2E). These results suggest that miR-574-3p plays an important role in tumor cell progression.

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Figure 2

Effect of miR-574-3p overexpression on PCa cell proliferation, migration and invasion in vitro and in vivo.

(A) miR-574-3p expression levels in PCa cell lines (PC3 and DU145) were determined by real-time PCR at 72 hours after transfection of Pre-miR miRNA precursor. miR-574-3p expression was normalized to RNU48. Data are presented as the mean ± SE. (B) Overexpression of miR-574-3p significantly inhibits cell viability. Cell viability was analyzed by the MTS cell proliferation assay 1, 2 and 4 days after transient transfection. *, P<0.05. (C) Over-expression of miR-574-3p significantly inhibits cell migration. After transfection (48 hours), a wound was formed by scraping and measured after 6, 12 and 24 hours. Representative images of wound healing assay are shown at 200× magnification. **, P<0.0001. *, P<0.005. (D) Over-expression of miR-574-3p significantly decreased cell invasion. Representative images of invasion assay are shown at 200× magnification. *, P<0.005. (E) Representative images of tumors in nude mice 5 weeks after subcutaneous injection of transfected miR-574-3p DU145 cell lines or control cell lines and time course of tumor growth.

miR-574-3p Influences Cellular Apoptosis in PCa Cells

Since miR-574-3p restoration significantly inhibited cell proliferation in PCa cell lines, we hypothesized that its restoration may induce apoptosis. Fig. 3A and 3B showed that the apoptotic and early apoptotic fractions (upper right and lower right in the quadrant images, respectively) were greater in miR-574-3p transfectants compared to control. This points to a pro-apoptotic role for miR-574-3p and suggests that it affects apoptotic pathways and regulates tumorigenicity. Therefore we examined the expression of various apoptotic proteins by Western blot analysis and found that miR-574-3p re-expression causes Bcl-xL down-regulation (Fig. 3C). Also, cleaved caspases-9 and -3 were up-regulated (Fig. 3C), further supporting the fact that miR-574-3p is a pro-apoptotic miRNA.

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Figure 3

Effect of miR-574-3p overexpression on apoptosis.

(A) Apoptosis assay using flow cytometry. Representative quadrant figures of miR-control and miR-574-3p transfectants in PC3 (upper) and DU145 (lower) cells. (B) The bar chart indicates the ratio of apoptotic cell fractions (early apoptotic plus apoptotic cells) in miR-574-3p transfectants compared with controls. Data for the apoptotic cell fractions are expressed as the relative value for the average expression of the miR-control transfectant. *, P<0.05. (C) Immunoblots analysis for apoptotic markers in miR-control and miR-574-3p transfected DU145 cells. GAPDH was used as a loading control.

Search for miR-574-3p Target Genes by in silico Analysis

To search for putative target genes of miR-574-3p, we used the TargetScan database. This program showed that miR-574-3p has 437 predicted target genes. We used in silico analysis to identify the biological processes or pathways potentially regulated by miR-574-3p. The candidate target genes were assigned to pathways using GeneCodis software analysis (http://genecodis.cnb.csic.es) [19], [20], [21], and statistically enriched pathways were identified such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’ (P<0.05, Table 3). We then performed gene expression analyses for all candidate target genes involved in each of the 9 pathways using microarray expression data, which were approved by the Gene Expression Omnibus (GEO) and focused on the ‘Pathways in cancer’. From this analysis, several crucial gene targets were identified, including tropomyosin 3 (TPM3), wingless-type MMTV integration site family, member 5A (WNT5A), ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC1), epidermal growth factor receptor (EGFR), retinoid X receptor, alpha (RXRA), v-akt murine thymoma viral oncogene homolog 2 (AKT2), and E1A binding protein p300 (EP300).

Luciferase Reporter Assays Using Vectors Containing 3′UTR Binding Sites of Putative Target Genes

To confirm the binding of miR-574-3p to the 3′ UTR of these 7 target genes, we performed luciferase reporter assays. Each target has one predicted binding site for miR-574-3p (Fig. 4A). We cloned the putative miR-574-3p 3′UTRs target into a luciferase reporter assay vector. Luciferase reporter assays demonstrated that miR-574-3p decreased the relative luciferase activities of RAC1, EGFR and EP300 (Fig. 4B) except for other four genes. Mutation of the putative miR-574-3p binding sites in these 3′UTRs decreased the response to miR-574-3p indicating that miR-574-3p binds directly to the 3′UTRs of RAC1, EGFR and EP300.

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Figure 4

miR-574-3p targets RAC1, EGFR, EP300.

(A) Putative miR-574-3p binding and mutated sites in the 3′UTR of target genes. (B) Luciferase reporter assays using vectors encoding putative 3′UTR binding sites. PC3 and DU145 cells were transiently transfected with Pre-miR miRNA precursor or negative control, followed by transient transfection with basic vector or wild-type 3′UTR reporter plasmids or mutated 3′UTR plasmids for 24 hours. 3′UTR reporter activity was measured by luciferase assay and normalized to the activity of Renilla luciferase. Data are presented as the mean ± SE. *, P<0.05. (C). The mRNA levels of the three target genes of miR-574-3p were determined by quantitative real-time PCR analyses after transfection with miR-574-3p mimics and negative control in PCa cell lines (PC3 and DU145). *, P<0.05. (D) Immunoblot analysis for target genes in miR-control and miR-574-3p transfected PC3 cells. GAPDH was used as a loading control.

Regulation of Target Gene Expression in PCa Cell Lines by miR-574-3p

Quantitative real-time PCR analysis showed that the mRNA expression levels of the three target genes in PC3 and DU145 were repressed in the miR-574-3p transfectants compared with the controls (Fig. 4C). The protein expression levels of the three target genes were also decreased in the miR-574-3p transfectants compared with the controls (Fig. 4D).

Cell Culture

Human PCa cell lines, PC3 and DU145 and a non-malignant epithelial prostate cell line, RWPE-1, were purchased from The American Type Culture Collection (Manassas, VA, USA). PCa cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. RWPE-1 cells were cultured in keratinocyte growth medium supplemented with 5 ng/mL human recombinant epidermal growth factor and 0.05 mg/mL bovine pituitary extract (Invitrogen, Carlsbad, CA, USA). Subconfluent cells (60%–70% confluent) were treated with genistein (25 µmol/L and 50 µmol/L; Sigma, St Louis, MO, USA) dissolved in dimethylsulfoxide and cells treated with vehicle (dimethylsulfoxide) served as control. Media and genistein were changed every day and cells were grown for 4 days.

RNA Extraction

RNA was extracted from FFPE human samples using a miRNeasy formalin-fixed paraffin-embedded kit (Qiagen, Valencia, CA, USA) after microdissection. To digest DNA, the Qiagen RNase-Free DNase kit was used. Total RNA was also extracted from PCa cell lines and a non-malignant epithelial prostate cell line using a miRNeasy mini kit (Qiagen) according to the manufacturer’s instructions.

microRNA Microarray

For miRNA microarray, total RNA was extracted from PC3 cells treated with genistein using a miRNeasy Mini Kit. The miRNA microarray analysis was carried out and analyzed by a commercial company (Phalanx Biotech, Belmont, CA, USA) using the human v3 miRNA OneArray platform that is designed to contain 100% of miRBase Sequence Database Release 17.0.

Quantitative Real-time PCR

Extracted total RNA was reverse transcribed into single-stranded cDNA using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR analysis was performed with an Applied Biosystems Prism7500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacturer’s protocol (Applied Biosystems). Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems). PCR parameters for cycling were as follows: 95°C for 20 seconds, 40 cycles of PCR at 95°C for 3 seconds, and 60°C for 30 seconds. All reactions were done in a 10-µL reaction volume in triplicate. The data were analyzed with the delta-delta Ct method to calculate the fold-change. TaqMan probes and primers for RAC1 (assay ID: Hs01902432_s1), EGFR (assay ID: Hs01076078_m1), EP300 (assay ID: Hs00914223_m1), GAPDH (assay ID: Hs02758991_g1), miR-574-3p (assay ID: 002349), RNU48 (Assay ID: 001006) were obtained from Applied Biosystems. GAPDH and RNU48 were used as internal controls.

Western Analysis

At 72 hours after transfection, cells were lysed with RIPA buffer (Pierce, Brebieres, France) containing protease inhibitors (Sigma). Protein quantification was done using a BCA protein assay kit (Pierce). Protein lysate (30 µg) was separated on 4% to 20% SDS polyacrylamide gels and transferred to a PVDF membrane. Antibodies to EP300 and Bcl-xL were purchased from Invitrogen and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against RAC1, EGFR and GAPDH were purchased from GeneTex (Irvine, CA, USA) respectively. Antibodies to cleaved caspase-3, -9 and caspase-3, -9 were purchased from Cell Signaling Technology (Danvers, MA, USA). After incubation with primary antibody the membrane was washed and then incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology, Danvers, MA, USA). Specific complexes were visualized with an echochemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, UK). The membrane was stripped using ReBlot Plus Strong Antibody Stripping Solution (Millipore, Billerica, MA, USA). The expression level of genes was then evaluated by using ImageJ software (ver. 1.43; http://rsbweb.nih.gov/ij/index.html).

Transfection

Pre-miR miRNA precursor and negative control (Applied Biosystems) were used in the gain-of-function experiments. RAC1, EGFR and EP300 siRNA (Sigma) and negative control siRNA (D-001810-10; Thermo Fisher Scientific, Waltham, MA, USA) were used in the loss-of-function experiments. PC3 and DU145 cells were transiently transfected using Lipofectamine 2000 transfection reagent (Invitrogen), according to the manufacturer’s recommendations.

Cell Proliferation, Migration, and Invasion Assays

Cell proliferation was measured using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI, USA) performed according to the manufacturer’s instructions. Cell proliferation was determined by absorbance measurements at 490 nm using SpectraMAX 190 (Molecular Devices Co., Sunnyvale, CA, USA). Cell migration activity was evaluated by a wound-healing assay. Cells were plated in six-well dishes, and the cell monolayers were scraped using a P-20 micropipette tip. The width of the initial gap (0 h) and the residual gap 6, 12 and 24 hours after wounding were calculated from photomicrographs. A cell invasion assay was carried out using modified Boyden Chambers consisting of transwell-precoated Matrigel membrane filter inserts with eight micron pores in 24-well tissue culture plates (BD Biosciences, Bedford, MA, USA). Minimum essential medium containing 10% FBS in the lower chamber served as the chemoattractant, as described previously [55]. All experiments were performed in triplicate.

In vivo Tumor Growth

All animal care was in accordance with the guidelines of the San Francisco Veterans Affairs Medical Center and the study was approved by the San Francisco VA IACUC (Protocol number: 11-008-01). Animal users have completed training programs to handle and work with mice through AALAS (American Association for Laboratory Animal Science) prior to animal experiments. For the subcutaneous xenograft mouse model, DU145 cells (2.5×10⁶) that were transiently transfected with miR-574-3p or miR-control were suspended in 50 µL RPMI 1640 medium and were subcutaneously injected into female nude mice (strain BALB/c nu/nu; Charles River Laboratories, Inc., Wilmington, MA, USA, 5 weeks old). A total of 8 nude mice (4-miR-574-3p, 4-miR-control) were used and tumor growth was examined over the course of 35 days. Tumor volume was calculated on the basis of width (x) and length (y): x²y/2, where x<y.

Apoptosis Assays

Fluorescence-activated cell-sorting (FACS) analysis for apoptosis was done 96 hours post-transfection, using Annexin V-FITC/7-AAD Kit (Beckman Coulter, Brea, CA, USA), according to the manufacturer's protocol. Stained cells were immediately analyzed with a flow cytometer (Cell Lab Quanta SC; Beckman Coulter).

Identification of miR-574-3p Regulated Target Genes and Bioinformatic Analysis

To search for genes regulated by miR-574-3p, we used TargetScan algorism (release 6.2, http://www.targetscan.org/). To identify the biological processes or pathways potentially regulated by miR-574-3p, we performed GeneCodis analysis (http://genecodis.dacya.ucm.es/) using all of the candidate genes. Then, to identify networks among the miRNAs and their target genes, we analyzed and characterized those genes in KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway categories. These data were used to examine miRNA-regulated molecular networks in human cells. We performed gene expression analyses of all candidate genes involved in each of the pathways using microarray expression data approved by the GEO and were assigned GEO accession numbers (GSE29079). In the Affymetrix Human Exon 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) datasets, we examined 47 PCa tissues and 47 normal prostate tissues, all of which were collected from patients who had not been exposed to neo-adjuvant radio-, cytotoxic- or endocrine therapy before the operation. The data was normalized and analyzed with the GeneSpring (Agilent Technologies, Santa Clara, CA, USA). Statistical analyses were conducted using the Mann Whitney U-test with cut-off P<0.05.

Plasmid Construction and Dual-luciferase Reporter Assays

For 3′ UTR luciferase reporter assay, PmirGLO Dual-Luciferase miRNA Target Expression Vector was used (Promega). The oligonucleotide sequences (wild-type) used are shown in Table S1. We also constructed mutated oligonucleotides for each of the wild-type oligonucleotides (Table S1). In a total volume of 25 µl, 1 µl each of 100 µM forward and reverse oligonucleotide, 2.5 µl of 10× annealing buffer (100 mM Tris–HCl, pH 7.5, 1 M NaCl and 10 mM ethylenediaminetetraacetic acid) and 20.5 µl water were incubated at 95°C for 3 min and then placed at 37°C for 15 min. The oligonucleotides were ligated into the PmeI–XbaI site of pmirGLO Dual-Luciferase miRNA Target Expression Vector. For 3′ UTR luciferase assay, PCa cells were co-transfected with Pre-miR miRNA precursor and pmirGLO Dual-Luciferase miRNA Target Expression Vectors using Lipofectamine 2000 (Invitrogen) and X-tremeGENE HP DNA Transfection Reagent (Roche Diagnosis, Basel, Switzerland, USA) according to the manufacturer’s instructions. Luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega) 24 hours after transfection. Firefly luciferase activities were normalized with Renilla luciferase. We also included basic vector containing no insert as a mock control.

We thank Dr. Roger Erickson for his support and assistance with the preparation of the manuscript.