Human prostate cancer cell lines, PC-3 and DU-145, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown in Dulbeccos modified Eagles medium (DMEM; Gibco^® by Life Technologies™, Grand Island, NY, USA) supplemented with 10% v/v inactivated fetal bovine serum (FBS; Biological Industries Israel Beit Haemek, Ltd., Kibbutz Beit-Haemek, Israel) and 100 U/ml penicillin + 100 µg/ml streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO₂.

Cells were plated at 1×10⁵ in a 6-well plate. Twelve hours later, cells were transfected with 50 nM of scrambled control, miR-194, miR-194 inhibitor, inhibitor control or siRNA for hNUDC (Suzhou GenePharma Co., Ltd., Suzhou, China) with the use of Lipofectamine^® 2000 reagent (Invitrogen by Life Technologies, Carlsbad, CA, USA). siRNA for hNUDC used here has been previously described (27). After transfection, the cells were processed for western blot analysis, quantitative real-time PCR (RT-qPCR), migration and invasion assays.

Total RNA was isolated from the prostate cell lines using TRIzol reagent, and quantified spectrophotometrically. The reverse transcription of miRNAs from total RNA (1 µg) was performed with miR-194-specific stem-loop primer (5-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCCACATG-3) using a Bestar™ qPCR RT kit (DBI^® Bioscience, Ludwigshafen, Germany). To measure hNUDC mRNA expression levels, the first strand of cDNA was synthesized using oligo (dT) primers by Bestar™ qPCR RT kit and the reverse transcription product was amplified using Bestar™ SybrGreen qPCR Mastermix (DBI^® Bioscience). The sequences of the primers specific for hNUDC were 5-CAGTGGGGTCTTGCTGTCATCT-3′ (forward) and 5-CTAACCCTTGCCTTTCAACTCA-3′ (reverse). Quantitative PCR was performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). The PCR reaction consisted in a denaturation step at 95°C for 10 min, followed by 40 amplification cycles at 95°C for 10 sec, and annealing at 60°C for 35 sec and extension at 72°C for 30 sec. Gene expression was normalized using endogenous β-actin or U6 as a control. Densitometric quantification was perfomed with the comparative cycle threshold method (2^−ΔΔCt) (36).

Total lysates from DU-145 and PC-3 cells (10 µg) were applied to 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% BSA in 1X Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. Western blot analysis was performed by incubating the membranes with primary antibodies for hNUDC (Abcam, Cambridge, MA, USA) at a dilution of 1:3,000 in 5% BSA in 1X TBST. After overnight incubation at 4°C, the membranes were incubated with goat anti-mouse IgG alkaline phosphatase (AP) conjugated secondary antibodies (Cell Signaling Technology, Inc.) at 1:1,000 for 1 h at room temperature. Signals were visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL, USA). Densitometric quantification was performed using ImageJ software.

3′UTR mRNA sequence of hNUDC containing a miR-194 binding site was amplified by the following primers: 5′-ATTACGCGTCCCCTGTTTTTTCCTCCCTG-3′ (forward) and 5′-CGGAAGCTTAGCTGGGCAAATCGTTTTAA-3′ (reverse). The PCR product was then cloned downstream to the luciferase gene in plasmid pMIR-Report. A mutation construct was made by deletion of several bases within the binding site and cloned into the same vector. The control vector was used for normalization of cell number and transfection efficiency. Co-transfection of synthetic miR-194 and luciferase report constructs were transfected in HEK-293T cells by using Lipofectamine 2000 reagent. Luciferase assays were performed using the Dual-Luciferase^® Reporter Assay System kit (Promega Corp., Madison, WI, USA) and luminescence was measured on a GloMax^® 96 microplate luminometer (Promega).

Cellular invasion and migration were assessed using the Cultrex^® Basement Membrane Extract (BME), PathClear^® (Trevigen, Gaithersburg, MD, USA) according to the manufacturers protocol. Cells at a density of 5×10⁴ were seeded onto Transwell^® Permeable Supports (Corning Incorporated, Corning, NY, USA). For the migration assay, the plates were incubated for 12 h at 37°C. The cells that migrated through the pores without the matrix to the lower surface of the membrane were fixed in 70% ethanol and stained with crystal violet. For the invasion assay, the plates were incubated for 48 h and the cells that invaded through the pores covered with the matrix to the lower surface of the membrane were fixed and stained. The cells were counted under a light microscope in five separate fields. Each blue point indicated an individual cell and all the images were scanned and counted at ×40 magnification.

Cells were transfected for 48 h and fixed with methanol prior to being stained with May-Grünwald-Giemsa staining solution for 15 min at room temperature. The stained slides were examined by a phase contrast microscope at ×40 magnification.

The packaged lentivirus expressing miR-194, anti-miR-194-sponge and GFP-control were purchased directly from ViGene Biosciences (Shandong, China). Stable expression of miR-194, GFP-control and anti-miR-194-sponge in PC-3 and DU-145 cells was achieved through selection with 2 µg/ml puromycin (Amresco, Solon, OH, USA).

The clonogenic cell growth of PC-3 and DU-145 cell lines was examined on soft agar using Cell Transformation Detection Assay (Millipore, Temecula, CA, USA). Briefly, each well of a 24-well plate was first layered with 0.8% agarose in growth medium (DMEM supplemented with 10% FBS). The cell lines to be tested were trypsinized, and 1×10⁴ cells were resuspended in a growth medium containing 0.4% agarose and then they were poured as a top layer in the 24-well plates. The plates were incubated at 37°C for 28 days. Colony formation was observed using Cell Stain Solution overnight and the diameter of colonies was scored under a microscope at ×40 magnification. Cells were counted with the Cell Quantification Solution after a 4-h incubation at 37°C, followed by a spectrophotometer reading at OD₄₉₀.

The female BALB/c nude mice (18 g, aged 6 weeks) were obtained from Guangdong Medical Laboratory Animal Center (permit no. SCXK 2013-0002). Animals were maintained and cared for in the Traditional Chinese Medicine and Marine Drugs Laboratory of Sun Yat-Sen University (permit no. SYXK 2014-0020) which conformed to the Guide for the Care and Use of Laboratory Animals. In vivo experiments were performed in accordance with a protocol approved by the Animal Ethics and Welfare Committee of Sun Yat-Sen University. PC-3 and DU-145 cell lines stably expressing miR-194, anti-miR-194-sponge or GFP-control were suspended in a PBS buffer at a concentration of 1×10⁷ cells/ml. Cell suspension (2×10⁶ cells/mouse) were injected subcutaneously below the right scapula of the BALB/c-nude mice. Seven mice from each of the three groups were used. The experiment ended when the tumor volume reached 2,000 mm³. The tumors were measured with microcalipers three times a week and the volumes were calculated as V (mm³) = length (mm) × (width (mm))² × 0.5. The mice were euthanized on day 30 and the tumors were harvested and weighed.

Statistical relevant differences were assigned to a p-value of at least ≤0.05 using an unpaired Students t-test in all functional assays and densitometric analyses of western blot analysis assays. All data represent at least three independent experiments. Results are expressed as the mean ± standard error (SE).

To determine whether hNUDC can be regulated by miRNAs, we used TargetScan (37,38) (http://www.targetscan.org/) and miRanda (39–41) (http://www.microrna.org/) to predict miRNAs that could potentially target the hNUDC 3UTR. A potential binding site for miR-194 was identified at the hNUDC 3UTR from nucleotides 1309–1316 (Fig. 1A). To validate hNUDC as a direct target of miR-194, both wild-type and mutant hNUDC 3UTRs were cloned downstream of the firefly luciferase ORF in a pMIR-Report vector (Fig. 1A). The wild-type and mutant hNUDC 3UTRs luciferase expression vectors were co-transfected with scrambled control or miR-194 mimic into HEK-293T cells. Relative luciferase activity was significantly reduced for wild-type hNUDC 3UTR, indicating that hNUDC is a potential direct target of miR-194 (Fig. 1B). Mutation in the predicted miR-194 target site abrogated inhibition by miR-194 mimic, confirming the functionality of this target site (Fig. 1B).

To determine if the hNUDC gene was a biologically relevant target of miR-194 in PCa cells, PC-3 and DU-145 cell lines were transfected with miR-194 mimic or miR-194 inhibitor. To demonstrate the specificity of miR-194 in order to evaluate the magnitude of the changes in hNUDC mRNA levels, non-targeting miRNA inhibitor and non-targeting scrambled miRNA were used as controls. qRT-PCR analyses revealed a significant reduction in the RNA expression of hNUDC following transfection with miR-194 mimic, whereas miR-194 inhibitor increased hNUDC levels (Fig. 2A). Western blot analysis was also employed to examine the hNUDC protein, showing a prominent decrease in the expression of hNUDC when PC-3 and DU-145 cells were transfected with miR-194 mimic. Conversely, inhibiting the endogenous miR-194 in PC-3 and DU-145 cells using miR-194 inhibitor increased the hNUDC protein level (Fig. 2B).

To date, migration and invasion are known as the key processes in many cancers. In order to investigate whether the ectopic overexpression of miR-194 is involved in these processes, we assessed the in vitro migratory and invasive capacities by Transwell migration assay. The ectopic expression of miR-194 decreased the invasiveness of PC-3 and DU-145 cells compared to the control mimic-transfected cells (Fig. 3A). Upon inhibition of endogenous miR-194 by miR-194 inhibitor, a visible opposite result was seen than in the control inhibitor transfected cells (Fig. 3A). Microscopy results revealed that upon transfection of miR-194 mimic, the loss of migratory capability was accompanied by a loss of cell elongation (Fig. 3B). These findings indicate that upregulation of miR-194 inhibits cell invasion and migration.

Since hNUDC is considered an important mediator of cell proliferation and cytokinesis, thus, it is expected that the inhibition of hNUDC would result in the failure of cell growth and give rise to cells with multiple nuclei. However, our cell proliferation and cell cycle analyses revealed that transfection of the miR-194 mimics into two PC-3 and DU-145 cell lines showed no influence on cell proliferation and cell cycle compared to the scrambled control (data not shown). Morphologically, transfection of cells with the miR-194 resulted in an increase in multinucleated cells coupled with abnormally large, flattened and accumulated multiple nuclei, whereas cells transfected with the mimic and inhibitor controls had normal nuclear morphology (Fig. 4A and B).

The aforementioned results demonstrated that miR-194 is a potent suppressor of cell migration and invasion through the targeting of hNUDC mRNA. Thus, we reasoned that siRNA-mediated knockdown of hNUDC should result in a cell phenotype similar to that of miR-194 overexpression. Indeed, by using hNUDC siRNA we were able to obtain a decrease in the expression of the hNUDC gene, reducing mRNA levels by 3.3-fold in the PC-3 cells and by 2.5-fold in the DU-145 cells, as determined by qRT-PCR analysis 48 h post-transfection (Fig. 5A). This result was confirmed by a significant reduction in hNUDC at the protein level (Fig. 5B). Similarly, miR-194 overexpression, significantly reduced the migration and invasion potential following knockdown of hNUDC expression (Fig. 5C and D). Following transfection with hNUDC siRNA, we observed that the multinucleated cells were enhanced following inhibition of endogenous hNUDC (Fig. 5E and F). These results strongly suggest that downregulation of hNUDC expression promotes invasion and migration of prostate cancer cells.

To examine whether miR-194 regulates primary tumor growth, a standard colony formation assay was used for these studies. We overexpressed miR-194 in PC-3 and DU-145 cells by transduction with high-titer lentivirus expressing miR-194 or anti-miR194-sponge using an empty vector as a control. An ~700-fold increase of miR-194 expression was detected by qRT-PCR in the transduced cells, in comparison to cells that were transduced with a control vector expressing GFP (Fig. 6A). As expected, a small decrease in miR-194 in the lentiviral expressing anti-miR-194-sponge was observed (Fig. 6A). The expression level of hNUDC protein was significantly decreased in response to ectopic miR-194 expression (Fig. 6B). Overexpression of miR-194 decreased the total number of cells observed when compared with the control (Fig. 6C). In contrast, anti-miR-194-sponge did not suppress cell growth in soft agar (Fig. 6C). Moreover PC-3 and DU-145 cells transfected with miR-194 formed smaller colonies compared to larger disseminated colonies formed by the GFP-control and anti-miR-194-sponge transduced cells after 30 days (Fig. 6D). The significant reduction of colonies suggests that miR-194 exhibits a tumor-suppressive function in cancer cells.

We next examined the effect of ectopic miR-194 expression on prostate cancer tumorigenesis in vivo. DU-145 cells stably expressing either miR-194 or anti-miR-194-sponge was subcutaneously injected into 6-week-old nude mice. The control group was injected with DU-145 stably expressing GFP. Tumor growth was monitored twice weekly for 30 days. As shown in Fig. 7, tumors derived from cells expressing anti-miR-194-sponge grew more rapidly. By the end of the study, the tumors were larger (tumor volume) and heavier (tumor weight) than the GFP-control. In contrast, tumors derived from the cells with miR-194 overexpression appeared significantly smaller than the tumors formed from the cells transduced with the GFP-control.