Background
Metastatic progression of prostate cancer is a major cause of death among men in the United States [
1]. Though cancer metastasis is a highly complex multi-step process facilitated by several key events and molecular players, the most effective way known to prevent this progression is by identifying and targeting the various genes involved in the process(es) [
2,
3]. Gene regulation is tightly controlled in the normal cells, thereby retaining the homeostatic expression of the appropriate genes for the functioning of the organism. Deregulation of these mechanisms in cancer causes the disrupted expression of the genes, which in turn furthers the cancer progression. MicroRNAs are a class of endogenous, small non-coding RNAs, 18 to 22 nucleotides long in their mature form, which can regulate a set of target genes and result in translational repression or mRNA degradation depending on the extent of complementarity and cellular context [
4,
5]. Recent studies have shown extensive dysregulation of miRNAs in prostate cancer [
6‐
8]. Many miRNAs have been implicated as tumor suppressors or oncomiRs depending on their target(s) and/or the global effects they have towards cancer progression [
9‐
12]. Though studies have been performed with respect to certain miRNAs and their specific targets in prostate cancer [
13,
14], not much is known about novel miRNAs targeting the players of cancer progression that can be used as diagnostic markers for early detection, or detection of a possible recurrence or resistance, or therapeutic agents to slow the progression. Identification of these novel miRNAs and their target gene(s), and the pathways they affect during cancer progression, will provide new insights into using them for diagnosis or determination of specific therapy regimens.
Migration and invasion enhancer 1 (
MIEN1), alternately called C17orf37, C35, RDX12, XPT4, ORB3 or MGC14832, is located in the 17q12-21 region of the human chromosome next to HER2/
neu in a tail-to-tail arrangement. MIEN1 is abundantly expressed in different stages and grades of prostate cancer phenotypes when compared to normal cells and tissues [
15]. MIEN1 has also been predicted as a novel breast cancer biomarker with increased expression in patients with metastatic progression to lung and liver, suggesting its importance in cancer metastasis [
16]. MIEN1 plays a role in prostate cancer migration and invasion through enhancement of filopodia formation by facilitating actin cytoskeletal rearrangement and by up-regulating the Akt dependent NF-κB target genes [
15,
17]. This was further confirmed by the recent determination of the solution structure of MIEN1 which predicts that Akt phosphorylation via MIEN1 may be dependent on the active redox-like motif in the MIEN1 structure [
18]. MIEN1 is also post-translationally modified by prenylation, via GGTase-I, at its C-terminus CVIL motif. Deletion of the motif not only led to the disruption of MIEN1 membrane localization and reduced invasive and migratory potential but also decreased metastasis to the lungs [
17]. Although abrogation of prenylation is a possible targeting strategy, it cannot be effectively used since it has been proven that many proteins involved in the regular functioning of the cell are prenylated, rendering this a very important modification. Hence, inhibition of prenylation could negatively impact multiple cellular processes [
19]. On the contrary, since MIEN1 is differentially expressed between normal and cancer cells and tissues, deciphering the regulatory mechanism(s) that explain the aberrant expression of MIEN1 in cancer will enable targeting MIEN1 using mechanisms that are endogenously prevalent thus forming an intervention for prostate cancer progression.
In this study, we have identified a novel miRNA, hsa-miR-940 (miR-940), which targets and regulates MIEN1 expression. Our study indicates that miR-940 expression inversely correlates with tumor progression in clinical prostate cancer and the loss of miR-940 in cancer causes an increased expression of MIEN1 which in turn enables prostate cancer progression. Ectopic expression of miR-940 resulted in not only decreased MIEN1 and its downstream effector molecules, but also reduced the migratory and invasive potential of the cells. Though the overall proliferation was unaltered, the ectopic expression of miR-940 reduced the anchorage-independent growth of cells, increased E-cadherin and decreased slug expression, suggesting facilitation of mesenchymal-to-epithelial transition (MET). Our results demonstrate that miR-940 may be a useful diagnostic marker as well as a therapeutic agent for prostate cancer.
Discussion
MIEN1, a novel gene in the 17q12 region of the human chromosome, is differentially expressed between cancer and normal cells and tissues [
16]. Previous studies indicate that MIEN1 plays an important role in prostate cancer progression [
15,
17]. In this study, we show that MIEN1 undergoes post–transcriptional regulation by miR-940. Our data show that miR-940 decreases the migratory and invasive potential of prostate cancer cells along with facilitation of MET. Also, decreased expression of miR-940 in prostate cancer specimens proves the clinical relevance of this miRNA, leading to our belief that miR-940 is a potential diagnostic marker and therapeutic agent.
The use of miRNAs for therapy and/or diagnosis of cancer is currently under consideration due to the accumulating evidence demonstrating their extensive deregulation in many cancers, including prostate cancer [
7,
28‐
30]. MiRNA mediated regulation of a target gene depends on multiple parameters including: 1) properties of miRNA responsive elements, such as the degree of complementarity and accessibility; 2) number of miRNAs that could target a single transcript; 3) expression of competitive endogenous mRNAs for a miRNA in a specific cellular context; 4) stimulus for the miRNA transcription/splicing and hence expression and stability; and, 5) other factors influencing the target mRNA stability and expression [
22‐
25]. Careful examination of the degree to which a gene is regulated by a miRNA and the overall effects of the miRNA mimic or inhibitor, are essential to determine the global role of the miRNA in any cellular context.
The distinct difference in the expression of MIEN1 and lack of protein despite mRNA expression in PC-3 cells directly implied involvement of post-transcriptional regulation. Our experiments confirmed that MIEN1 is indeed regulated by miRNA and led to the identification and validation of miR-940. Alterations in mRNA half-life in the presence of the miRNA elucidated the effect of miR-940 on MIEN1 mRNA stability. Furthermore, using luciferase assays, we ascertained that miR-940 binds directly to the 3′UTR of MIEN1 to cause its suppression.
MIEN1 is minimally expressed in several normal tissues compared to its overexpression in cancer [
16]. Its proximity to the HER2/
neu locus on chromosome 17 explains its frequent amplification (in 79% of breast cancers) with HER2 amplicon [
31]. A recent study using a set of eight genes, including MIEN1, revealed a moderate response to adjuvant Trastuzumab therapy even in HER2 negative breast cancer, confirming the importance of this gene in responses to neo-adjuvant therapies [
32]. Katz
et al. have shown that the overall survival of breast cancer patients is low in cases where MIEN1 is highly expressed while lower expression indicates better prognosis [
33]. It is well known that prostate cancer-related deaths are due to metastasis rather than the presence of a primary tumor alone. Metastasis is a complex process involving multiple intermediate steps, from detachment of the cells at the primary site to formation of secondary tumor. The tumor cells evade the resistances faced at every step by different mechanisms [
2,
3]. Our previous study has shown that cells overexpressing MIEN1 have a higher metastatic potential, though it does not mean quicker onset or initiation of the tumor [
17]. We have also previously shown that MIEN1 increases phosphorylation of Akt causing the translocation of NF-κB to the nucleus and then transcriptionally activating downstream effectors like MMP-9, uPA and VEGF [
15]. These proteases and angiogenic factors are known to cleave extracellular matrix, hence facilitating migratory and invasive potential of the cells. Taken together, these studies confirm that MIEN1 plays an important role in the progression of cancer rather than in the initiation of the tumor. Here, we show that upon ectopic reintroduction of miR-940, the mRNA as well as protein levels of the effector molecules decrease in DU-145. Conversely, downregulation of the miRNA using inhibitors increases the effector molecules in PWR-1E. Hence, miR-940, indirectly through MIEN1, is capable of decreasing expression levels of specific proteins that facilitate migration and invasion.
It is known that miRNAs can affect expression by causing mRNA degradation through complex formation with RNA-induced silencing complex or by repressing the translation of the mRNA, thus inhibiting formation of the protein [
22]. The degree of complementarity and the competitive endogenous mRNAs determine the fate of the miRNA-mRNA complex [
24]. Here, we see that miR-940 expression is highest in the immortalized PWR-1E cells, followed by PC-3 cells and lowest in DU-145. In addition to this expression pattern, our data prove that inhibition of miR-940 has different effects on MIEN1 mRNA and protein levels in the various cell lines. This implies that the inhibition of MIEN1 using miR-940 affects MIEN1 in a manner dependent on not only the cellular context (other competitive endogenous mRNA in the specific cells) but also on the endogenous miRNA levels. While in PWR-1E, the endogenous miR-940 potentially degraded MIEN1 mRNA; the overexpression of anti-miR-940 resulted in attenuation of MIEN1 mRNA degradation, thus causing an increase in both MIEN1 mRNA and protein levels. Conversely, the loss of the endogenous miR-940 in DU-145 possibly led to the overexpression of both MIEN1 mRNA and protein; and hence ectopic overexpression of the miR-940, as we have observed, caused MIEN1 mRNA degradation resulting in significant depletion of both transcript and protein levels. However, in PC-3, where MIEN1 mRNA is expressed but protein is low, inhibition of miR-940 with anti-miR-940 resulted in no further increase of MIEN1 mRNA but only increased MIEN1 protein. Additionally, the inhibition of endogenous MIEN1 mRNA with ectopic miR-940 in PC-3 decreased MIEN1 transcript. Together, this indicates that the endogenous miR-940 was causing translational repression of the MIEN1 mRNA rather than degradation in PC-3. Thus, these results show that while miR-940 causes MIEN1 mRNA degradation in DU-145 (ectopic and conceivably endogenous expression) and PWR-1E (endogenous), it causes translational repression of MIEN1 in PC-3 cells (endogenous). The unique ability of this miRNA to perform both mRNA degradation as well as translational repression of the same target depending on the levels of the miRNA and the cellular context seems like a novel finding.
MicroRNAs could target multiple transcripts, thus eliciting a response which is dependent on the combined effects on its targets [
24]. A recent study implied that miR-940 could be one of the regulators of alpha-1 antitrypsin [
34]. Loss of this serine proteinase inhibitor results in increased risk of lung and liver cancers while its elevated serum levels are associated with prostate cancer [
35], supporting our hypothesis that the miR-940 is lost in cancer cells and tissues. In our study, we performed experiments to determine the effects miR-940 would have on migration and invasion, thus delineating the mechanism by which miR-940 could affect cancer progression, based on its regulation of MIEN1, a validated player in the regulation of prostate cancer migration and invasion. We observed a decrease in both the migratory and invasive potential of the cells upon ectopic expression of the miRNA and the converse was seen when the miRNA was inhibited. Additionally, these effects were negated upon re-introduction of the non-targetable MIEN1 into the system. Hence, we are the first to report that miR-940 inhibits prostate cancer migration and invasion, at least in part via MIEN1 along with other probable targets. The ability of cells to form disseminated colonies without attaching to the substratum is very important to determine the tumorigenicity of the cells [
26,
27,
36]. Previous reports indicate that MIEN1 enhances EMT in breast cancer [
33]. Our study demonstrates that miR-940 completely inhibits this ability of prostate cancer cells along with promoting MET by increasing the E-cadherin and decreasing Vimentin expression. The decrease in slug, an indicator of cells losing their mesenchymal trait [
37], was also observed in corroboration. Since EMT is a very complex yet important process in prostate cancer progression [
38‐
40], further investigation to identify the exact mechanism by which miR-940 facilitates this transition is required. It is also important to determine the different global pathways and the proteins that may be altered by miR-940 that culminates in miR-940 mediated inhibition of prostate cancer progression. Since miR-940 is a very novel miRNA whose function has never been validated or reported in any pathway before, in our study we used a set of common genes predicted to be targets of miR-940 by multiple algorithms. The extensive gene list was then categorized into known and validated pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping and annotation table available in the Database for Annotation, Visualization and Integrated Discovery 6.7 (DAVID 6.7) [
41,
42]. The preliminary examination of the results obtained from DAVID was then represented as a function of the number of genes involved within the pathway that could be downregulated by miR-940 (Additional file
6: Table S1A), and further classified based on the significance of the overall pathway alteration (Additional file
6: Table S1B). Interestingly, the pathway with most number of genes affected was a global pathway in cancer. Also, many other predictions within the threshold set indicated the regulation of other pathways considered important in cancer. Thus, miR-940 may be eliciting the responses we have observed via other targets in addition to MIEN1 and this needs further validation.
Studies over the past decade have proven beyond question that in addition to understanding the regulation of pathways, early stage identification and targeting of prostate cancer is of primary importance in order to prevent metastasis of the disease. Use of deregulated miRNA profiles is currently under consideration to enable the advancement of detection and diagnoses [
10,
28‐
30]. We have seen that miR-940 expression is high in the normal and benign glands in tissues obtained from patients who have undergone prostatectomy compared to lower expression in the tumor. Associating the expression patterns of miR-940 and MIEN1 with the early detection and differentiation of the indolent from aggressive disease will be a valuable tool that could be used clinically for early prostate cancer detection. Use of circulating miRNAs from the serum is not only minimally invasive but also reliable since miRNAs are highly stable in the blood and hence can be used as potential biomarkers [
43,
44]. Apart from the potential use of miR-940 in tissues and serum as a biomarker along with the expression of MIEN1, studying the regulation of miR-940 itself may provide more insight into the mechanism of its expression pattern and the reasons for its loss in cancer, which from our results, indicates facilitation of cancer progression. Examination of miR-940 promoter exhibited high possibility of methylation and this warrants further investigation.
Materials and methods
Cell lines, cell culture, siRNA, miRNA and plasmid transfections
Human prostate carcinoma cells DU-145 (ATCC HTB-81), PC-3 (ATCC CRL-1435), and LNCaP (ATCC CRL-1740) were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (Life Technologies). Immortalized non-tumorigenic prostate epithelial cell line HPV-18C-1 (a kind gift from Dr. Jhong S. Rhim, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD) and PWR-1E (ATCC CRL-11611) were maintained in Keratinocyte-SFM (Life Technologies) supplemented with bovine pituitary extract (25 μg/ml) and recombinant epidermal growth factor (0.15 ng/ml). Cells were cultured at 37°C with 5% CO
2. The cell lines were authenticated according to “Authentication of Human Cell Lines: Standardization of STR Profiling” using
GenePrint® 10 System (Promega); all cell lines and their passages exhibited >80% match to the initial cell line STR profile provided by ATCC [
45]. The smart pool siRNAs were obtained from Dharmacon (Thermo Fisher Scientific), while the precursor and inhibitor miRNA oligos (Pre- and Anti-miR) were purchased from Ambion (Life Technologies). The final concentration of the miRNA oligos used for transfection was determined by preliminary concentration-dependent studies and remained constant for all the experiments. Plasmid transfections were performed using Lipofectamine 2000 while Lipofectamine RNAiMAX was used for RNAi transfections, performed according to the manufacturer’s protocols (Life Technologies).
Antibodies and reagents
The following antibodies and reagents were used: Mouse monoclonal and mouse polyclonal MIEN1 (Abnova; antibody specificity tested and proven in previous studies[
15,
17]), rabbit polyclonal MIEN1 (Life Technologies; antibody specificity tested in previous studies[
15]), mouse monoclonal GAPDH (Santa Cruz Biotechnology), rabbit monoclonal pNF-κB p65 S536 and rabbit polyclonal MMP-9 (Cell Signaling Technology), mouse monoclonal VEGF and uPA (R&D Systems), mouse monoclonal Alexa Fluor 594 conjugated Phalloidin (Life Technologies), mouse monoclonal E-cadherin (BD Biosciences), Vimentin (supernatant developed in mouse and tested against human antigen, Developmental Studies Hybridoma Bank), anti-mouse and anti-rabbit IgG (Promega), AlexaFluor 488 goat anti-mouse IgG and AlexaFluor 594 goat anti-mouse IgG (Life Technologies) sheep anti-DIG-AP antibody and NBT-BCIP ready-to-use tablets (Roche), sheep serum (Jackson ImmunoResearch), rabbit IgG, BSA, levamisole hydrochloride, Tris-HCl (pH 7.4), nuclease free water, SSC buffer, Xylene, Tween-20, Nuclear Fast Red, Hematoxylin and Eosin (Sigma-Aldrich) and Permount and PBS (Thermo Fisher Scientific).
In silico analyses were performed to determine the putative miRNAs that could target MIEN1. The software programs used included miRANDA [
20], PicTar [
46], miRBase [
47] and TargetScan [
21], all of which used the 3′UTR as the target region to determine miRNA recognition elements and provided scores to determine predictive values.
For microarray based hybridization, DU-145 and PWR-1E cells were trypsinized, spun down, washed with sterile PBS and frozen immediately at -80°C. The samples were de-identified and shipped to LC Sciences (Houston, TX) for microarray hybridization. In brief, total RNA was isolated from the cells and enriched for small RNA (<300nt). Subsequently, the small RNAs were 3′ extended with polyA tail and an oligonucleotide tag was ligated to it for fluorescent dye staining (Cy3). The samples were then hybridized to the probe set on the plate (probes consisted of sequences complementary to miRNA from miRBase as well as the specially requested custom probes). After hybridization, the miRNA expression was detected by fluorescence labeling using tag-specific dye. Images collected were analyzed using Array-Pro image analysis software. Data analysis involved subtraction of the background along with normalization. Paired t-test results were provided for further interpretation and study.
qPCR
Total RNA was isolated from the cell lines using TRIzol (Life Technologies) and quantified. Equal amount of RNA was used for the one-step or two-step qPCR performed using the Superscript III SYBR Green qRT-PCR kits, according to manufacturer’s instructions (Life Technologies). For miRNA, PCR was performed using NCode VILO miRNA cDNA Synthesis and EXPRESS SYBR GreenER miRNA qRT-PCR Kits (Life Technologies), according to the manufacturer’s protocol. The primers (sequences provided in the Supplementary materials and methods; Additional file
7) were designed using Primer 3 [
48] and synthesized by Integrated DNA Technologies (Coralville, IA). PCR was performed using Realplex
2 Mastercycler ep
gradient S thermal cycler (Eppendorf).
Western blotting
Western blotting was performed according to standard protocols. Briefly, total protein was isolated using NP-40 lysis buffer and estimated using the standard Micro BCA Protein Assay Kit (Pierce Biotechnology). NuPAGE® Novex® 4-12% Bis-Tris Gels were used and the samples were transferred onto nitrocellulose membranes using an iBlot (Life Technologies). Membranes were blocked in 5% non-fat dry milk or 1% BSA prior to antibody subjection. The chemiluminescent reaction was captured by the AlphaImager (ProteinSimple) and bands were analyzed using ImageJ software [
49].
Northern blotting
Northern blotting was performed using miRNA Northern Blot Assay Kit and custom ordered biotin-labeled miR-940 and U6 control probes (Signosis) with one microgram of total RNA from each cell line, according to manufacturer’s instructions.
RNA stability assay
Cells were transfected with the precursor oligomiRs and 48 hours after transfection, treated with 10 μg/ml Act-D (Sigma-Aldrich). RNA was isolated at several time points and quantified. Equal amounts of RNA were used to run qPCR to determine MIEN1 levels.
Luciferase reporter assay
Cells were transfected with 3′UTR luciferase constructs (Origene) - Empty Vector (Vec) or 3′UTR-MIEN1 (MIEN1WT / MIEN1Mut) and miR-940 or miR-NT in duplicate. Luciferase assay was performed using the Luciferase Assay System (Promega) according to manufacturer’s instructions and luminescence read using Synergy2 Alpha Microplate Reader (BioTek).
Migration assay
For migration assay, a scratch was made in a monolayer of transfected cells using a pipet tip, 48 hours after transfection. Fresh media was added immediately to remove the floating cells and the scratch and surrounding cells were imaged at T0 (immediately after scratching). Images were captured at specific time points from at least ten independent fields to determine the wound closure. Migration was calculated as a percentage of the area covered by the cells compared to the original wound area.
Invasion assay
Invasion assay was performed with transwell invasion assay inserts and 24-well plates (BD Biosciences) according to manufacturer’s protocol. In brief, cells were transfected with the miRNA oligomiRs and the inserts were coated with Matrigel (BD Biosciences). Cells were trypsinized 48 hours after transfection and 500 μl of the cell suspension (concentration of 5 X 104 cells/ml) was plated in duplicate in Matrigel-coated and non-coated transwell inserts with fetal bovine serum as a chemoattractant in the bottom well. The lower side of the transwell membranes were fixed and stained with 0.05% crystal violet 24 hours after plating. Fold change in invasion was calculated as a ratio of cells invading the Matrigel matrix-coated insert membrane to the cells migrating through the uncoated membrane. The invasion of no-targeting miRNA transfected cells was considered as 1 and the fold change was calculated accordingly.
Flow cytometry
DU-145 cells were transfected with miRNA mimics or siRNA against MIEN1 and subjected to cell cycle analysis using Propidium Iodide in a Beckman Coulter Cytomics FC 500 Flow Cytometer. In brief, transfected cells were trypsinized, washed with PBS, counted and resuspended to a concentration of 1.5 × 106cells/ml, 72 hours after transfection. Cells were fixed in cold ethanol at 4°C, overnight. After washing with PBS and centrifuging the suspension, the pellet was resuspended in PI with RNaseA and incubated at 4°C for about 3 hours in the dark before analysis.
Anchorage-dependent and -independent growth assays
For the anchorage-dependent clonal assay, cells were treated with precursor miR-NT/940 for 48 hours and seeded (2500 cells per well) on polystyrene coated 6-well plates. After 12 days, the colonies were fixed and stained with 0.05% crystal violet or subjected to immunofluorescence. Only individual colonies (>50 cells per colony) were considered to obtain the average number of colonies for each treatment.
For anchorage-independent colony formation assay, cells were treated with precursor miR-NT/940 for 48 hours before re-plating (5000 cells per ml per well) on soft agar (cells in 2X media:agar = 1:1). Colonies were stained with 0.05% crystal violet and counted after 12 days of incubation in soft agar.
Immunofluorescence and confocal microscopy
Cells were transfected, as described in anchorage-dependent assay, plated on coverslips to at least 90% confluence, fixed with 4% paraformaldehyde, permeabilized with 100% methanol, blocked with 1% BSA and stained for the specific proteins. The coverslips were mounted using PermaFluor Mountant (Thermo Fisher Scientific) and imaged using a Zeiss confocal microscope LSM 510 under 40X, water immersion objective. At least five independent fields per experiment were captured. The images were analyzed with LSM software and the predominant pattern is represented here.
In situ hybridization and immunohistochemistry
Archived paraffin-embedded prostate tumor with matched normal and tumor infiltrating normal gland tissue sections from multiple patients were collected under the approval of the Institutional Review Board at the site. The study protocol was approved by the Institutional Review Board at UNT Health Science Center. The anatomic pathologists independently read the slides and graded the Hematoxylin & Eosin (H&E) stained sections to provide scores (1-5; based on predominant primary Gleason pattern) and read the hybridized sections to determine miR-940 intensity scores (1-5; 1 being basal to very low to 5 being high intensity) for the matched normal and prostate progression sections; a chromogenic assay based on DIG labeled probes detected by alkaline phosphatase conjugated anti-DIG and NBT-BCIP substrate was used for miR-940 staining. The Exiqon (Denmark) miRCURY LNA™ microRNA ISH Optimization Kit (FFPE) was used to standardize and perform
in situ hybridization, using scrambled miRNA and the 5′- and 3′-DIG double labeled miR-940 probes. The extent of Proteinase-K treatment, the hybridization time and temperature, and incubation with the substrate were all standardized for the probes. Correspondingly, MIEN1 and isotype-specific rabbit IgG antibodies were used for immunohistochemistry that was performed on the serial sections according to standard protocols. The images were captured as described previously [
50]. ImageJ analysis of the staining intensities in the various tissues was performed with the “Colour Deconvolution” plugin [
49].
Statistical analyses
The results were represented as mean ± S.E.M of independent experiments. The p-value was calculated according to Student’s t-test when comparing two groups using GraphPad P-value calculator. Multiple groups were compared by one-way ANOVA, when necessary, followed by pair-wise comparisons with post-hoc test. The differences were considered significant if p-value was at least ≤0.05.
Consent
The patients provided consent for the use of the tissues for research and publication.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SR, SD and JKV conceptualized the study. SR designed, standardized and performed the assays, quantified the results and conducted statistical analysis. AVP and RJH acquired patient specimens and analyzed the H&E, in situ hybridization and immunohistochemistry staining as independent pathologists. SR and RKR performed cell line authentications. SR and JKV interpreted the data and prepared the manuscript. RJH and RKR critically reviewed the manuscript. JKV supervised the study. All authors read and approved the final manuscript.