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Keywords:

  • microRNA-205;
  • IL24;
  • IL32;
  • transcriptional activation;
  • prostate cancer

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

BACKGROUND:

MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of approximately 60% of all human genes. They play important roles in numerous cellular processes, including development, proliferation, and apoptosis. Currently, it is believed that miRNAs elicit their effect by silencing the expression of target genes. In this study, the authors demonstrated that miRNA-205 (miR-205) induced the expression the interleukin (IL) tumor suppressor genes IL24 and IL32 by targeting specific sites in their promoters.

METHODS:

The methods used in this study included transfection of small RNAs; quantitative real-time polymerase chain reaction; in situ hybridization; fluorescence-labeled in situ hybridization; cell cycle, apoptosis, cell viability, migratory, clonability, and invasion assays; immunoblotting; and luciferase reporter, nuclear run-on, and chromatin immunoprecipitation assays.

RESULTS:

The results revealed that miR-205 was silenced in prostate cancer. Its re-expression induced apoptosis and cell cycle arrest. It also impaired cell growth, migration, clonability, and invasiveness of prostate cancer cells. Micro-RNA-205 induced the expression of tumor suppressor genes IL24 and IL32 at both the messenger RNA and protein levels. The induction of in vitro transcription and enrichment of markers for transcriptionally active promoters in the IL24 and IL32 genes was observed in response to miR-205.

CONCLUSIONS:

In this study, a new function for miR-205 was identified that specifically activated tumor suppressor genes by targeting specific sites in their promoters. These results corroborate a newly identified function that miRNAs have in regulating gene expression at the transcriptional level. The specific activation of tumor suppressor genes (eg, IL24, IL32) or other dysregulated genes by miRNA may contribute to a novel therapeutic approach for the treatment of prostate cancer. Cancer 2010. © 2010 American Cancer Society.

MicroRNAs (miRNAs) are nonprotein-coding sequences that are believed to regulate the expression of up to 60% of human genes, either by inhibiting mRNA translation or by inducing its degradation.1, 2 These small RNAs play important roles in numerous cellular processes, including development, proliferation, and apoptosis.3 In addition to their crucial role in cellular differentiation and organism development,4 miRNAs frequently are misregulated in human cancers5, 6 and can act as either potent oncogenes or tumor suppressor genes.7

Prostate cancer (PCa) is the most frequently diagnosed malignant tumor and the second leading cause of cancer deaths among men in the United States. It is estimated that, in 2009, there will have been >192,280 newly diagnosed cases of PCa and >27,360 attributed deaths.8 Although surgery and radiotherapy generally are effective against clinically localized PCa, androgen ablation, the treatment of choice for advanced disease, leads to only temporary tumor regression.9 The lack of available treatment options for effectively eradicating advanced PCa makes the development of alternative approaches urgent. Understanding the molecular alterations that distinguish progressive disease from nonprogressive disease will help identify novel prognostic markers or therapeutic targets. Some aberrantly produced miRNAs have been identified in PCa cell lines, xenografts, and clinical samples.10 These miRNAs may play critical roles in the pathogenesis of PCa.

miRNA-205 (miR-205) expression in cancer is controversial, because it reportedly is either up-regulated11, 12 or down-regulated13, 14 in tumor tissues compared with normal tissues. Currently, it is believed that miRNAs elicit their effect by silencing the expression of target genes.15 Given the functional complexity of miRNA-mediated gene regulation, it is unlikely that the effects of these molecules are limited to gene silencing. The objective of the current study was to investigate the potential involvement of miR-205 in positively regulating the expression of the interleukin (IL) tumor suppressor genes IL24 and IL32.

It has been reported that IL24 is a novel tumor suppressor gene whose expression is lost during tumorigenesis.16 Gene transfer of an adenovirus-expressed melanoma differentiation associated 7 (mda-7)/IL24 into numerous histologic types of human tumor cell lines, including melanoma, glioblastoma, breast cancer, lung cancer, pancreatic cancer, and others, resulted in tumor-specific growth arrest.17-20 In addition to its direct apoptosis-inducing properties, IL24 has antiangiogenic, radiosensitizing, immunostimulatory, and potent “bystander” antitumor activity21-23 These divergent anticancer properties of mda-7/IL24 make it an ideal candidate for anticancer therapy. A recent phase 1 clinical trial in which a replication-incompetent adenovirus that expressed IL24 was used produced evidence of clinical activity with limited toxicity. Thus, IL24 is being hailed as a “magic bullet” for cancer gene therapy. IL32 is a novel cytokine that has been implicated in inflammation24 and cell death.25 It has been established that IL32 associates specifically with apoptotic T cells, and ectopic expression of IL32 in HeLa cells causes apoptosis.25 Our data indicated that there was strong induction of the IL24 and IL32 tumor suppressor genes in response to miR-205. In the current study, we identified a new function for miR-205 in regulating tumor suppressor genes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Experimental Procedures

Cell culture and microRNA transfection

Three human prostate carcinoma cell lines (LNCaP, PC3, and Du145) and a nonmalignant epithelial prostate cell line (RWPE-1) were obtained from the American Type Culture Collection (Manassas, Va). All cell lines were cultured as described previously.26 Transfection of miRNA was carried out by using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif) according to the manufactures's protocol. All miRNA treatments proceeded for 72 hours.

Quantitative real-time polymerases chain reaction

Mature miRNAs and other messenger RNAs (mRNAs) were assayed using TaqMan miRNA assays and gene expression assays, respectively, in accordance with the manufacturer's instructions (Applied Biosystems, Foster City, Calif). Samples were normalized to RNU48 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as indicated. Comparative real-time polymerase chain reaction (PCR) was performed in triplicate, including no-template controls.

In situ hybridization

For cell lines, the cells were grown on sterile slides in 100-mm Petri dishes for 48 hours. Then, these cells were stained by using digoxigenin (DIG)-labeled, locked nucleic acid (LNA)-based probes specific for miR-205 according to the manufacturer's protocol (www.exiqon.com accessed December 15, 2009). Last, the cells were detected by using anti-DIG-fluorescein, fragment antigen-binding (Fab) fragments (Roche Applied Science, Indianapolis, Ind). Nuclei were stained routinely by using 4′,6-diamidino-2-phenylindole (DAPI). Tissue array slides contained human normal adjacent prostate tissue samples (n = 15) and samples stage II (n = 14), stage III (n = 52), stage IV (n = 6), and metastatic (n = 8) carcinomas. Hybridizations were performed overnight at 52°C after the addition of 50 nM DIG-labeled, LNA-based probes that were specific for miR-205 and U6 (Exiqon Inc., Woburn, Mass). Alkaline phosphatase activity was detected using BM Purple AP Substrate (Roche Applied Science). In situ hybridization (ISH) results were graded semiquantitatively according to staining intensity, scored from 1 to 4, and normalized to U6 levels.

Immunoblot analysis

Immunoblot analysis was performed as described previously.27 Antibodies specific for p21WAF1 (catalog no. 05-345; Upstate Biotechnology, Billerica, Mass), p27 (catalog no. 2552; Cell Signaling Technology, Beverly Mass), IL24 (catalog no. ab56811; Abcam PLC, Cambridge, United Kingdom), IL32 (catalog no. ab62580; Abcam PLC), p38 (catalog no. 9212; Cell Signaling Technology), BCL2-antagonist/killer (BAK) (catalog no. 53251; AnaSpec Inc., Fremont, Calif), flagellar biosynthesis protein (FLIP) (catalog no. 3210; Cell Signaling Technology), BCL2-associated X protein (BAX) (catalog no. 2774; Cell Signaling Technology), BH3-interacting domain death agonist (BID) (catalog no. sc-11423; Santa Cruz Biotechnology, Santa Cruz, Calif), stress-activated protein-Jun N-terminal kinase (SAP-JNK) (catalog no. 9252; Cell Signaling Technology), and GAPDH (catalog no. 2118; Cell Signaling Technology) were used.

Flow cytometry, cell viability, migratory, clonability, and invasion assays

Fluorescence-activated cell sorting analyses were done 72 hours after transfection using the nuclear stain DAPI for cell cycle analysis or the annexin V-fluorescein isothiocyanate (FITC)/7-aminoactinomycin D (7-AAD) Kit (Beckman Coulter, Inc. Fullerton, Calif) for apoptosis analyses according to the manufacturer's protocol. Cell viability was determined at 24 hours, 48 hours, and 72 hours by using the CellTiter 96 AQueous 1 Solution Cell Proliferation Assay Kit (Promega, Madison, Wis) according to the manufacturer's protocol. For the colony formation assay, cells were seeded at low density (1000 or 200 cells per plate) and were allowed to grow for 3 weeks. Then, the cells were stained with crystal violet, and colonies were counted. An artificial “wound” was created on a confluent cell monolayer, and photomicrographs were taken after 24 hours for a migration assay. The invasiveness of PC3 cells was assessed with an invasion assay in a modified Boyden chamber as described elsewhere.28

Luciferase assays

A 2.2-kb promoter reporter vector of IL24 (identification no. (ID), 114318_CHR1_P1626_R1[IL24]) was obtained from SwitchGear Genomics (Menlo Park, Calif) along with the positive (ID, RPL10_PROM) and negative (ID, EMPTY_PROM) promoter control vectors. The assay was performed according to the manufacturer's protocol (www.switchgeargenomics.com accessed December 25, 2009).

Nuclear run-on assays

Nuclear run-on assays were performed as described by Kim et al.29 Metastatic PCa PC3 cells were transfected with control or miR-205 duplexes (Ambion Inc., Austin, Tex) at 50 nM final concentration using Lipofectamine 2000 (Invitrogen). Seventy-two hours after transfection, 2 × 107 cells were washed with cold phosphate-buffered saline, harvested, lysed on ice in 0.5% Nonidet P-40 lysis buffer (10 mM Tris-HCl, pH 7.4; 10 mM NaCl; and 3 mM MgCl2), and centrifuged at ×500g for 10 minutes. Supernatants were removed, and the nuclei were incubated in reaction buffer (10 mM Tris-HCl, pH 8.0; 5 mM MgCl2; and 0.3 mM KCl) and 2.5 mM nucleoside triphosphate plus biotin-16-uridine 5′-triphosphate mix (Roche Applied Science, Indianapolis, Ind) for 45 minutes at 30°C. The transcription reaction was stopped by adding RNA STAT-60 Reagent (Tel-Test, Ind., Friendswood, Tex) to isolate total nuclear RNA according to manufacturer-recommended protocols. Biotinylated nascent RNA transcripts were isolated by incubation with streptavidin beads (Active Motif, Carlsbad, Calif) for 2 hours at room temperature on a rocking platform. Beads were collected by centrifugation and washed once with 2 × standard saline citrate (SSC)-15% formamide for 10 minutes and twice with 2 × SSC for 5 minutes on a rocking platform. Biotinylated RNA was eluted from streptavidin beads in H2O or 10 mM ethylene diamine tetracetic acid, pH 8.2, by incubation at 90°C for 10 minutes and analyzed by quantitative real-time PCR (qRT-PCR).

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assays were performed using the EZ-ChIP Kit (Upstate Biotechnology) as described previously.27 Immunoprecipitation was performed using antibodies purchased from Upstate Biotechnology and recognized acetyl histone 3 (catalog no. 06-599), acetyl histone 4 (catalog no. 06-866), and dimethyl-histone H3 lysine (Lys) 4 (catalog no. 07-030). Power SYBR Green PCR Mastermix (Applied Biosystems) was used to perform real-time PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems). Signals were also confirmed by conventional PCR and gel analyses.

Statistical Analysis

Statistical analysis was performed using StatView version 5.0 for Windows (Stata Corp., College Station, Tex). The Student t test was used to compare the different groups. P values <.05 were regarded as statistically significant and are represented by asterisks in the figures.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

MicroRNA-205 Is Down-Regulated in PCa Cell Lines and Tissue Specimens

Quantitative real-time PCR revealed that miR-205 was either undetectable or highly repressed in both androgen-dependent (LNCaP) and androgen-independent (PC3, Du145) PCa cells compared with nonmalignant RWPE-1 cells (Fig. 1A). Because it has been reported that LNA-modified miRNA probes markedly increase hybridization affinity to miRNAs compared with traditional RNA-based or DNA-based probes, a digoxigenin (DIG)-labeled LNA-miR-205 probe was used to detect miR-205 abundance in human PCa cell lines using ISH and an FITC-labeled anti-DIG antibody. Consistent with the results described above, fluorescent signal was detected in RWPE-1 cells and represented the abundance of miR-205 in this cell line, whereas the signal was absent in the cancer cell lines LNCaP, PC3, and Du145 (Fig. 1B).

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Figure 1. MicroRNA-205 (miR-205) is silenced in the human prostate cancer cell lines PC3, LNCAP, and Dul45 and also was evaluated in a nonmalignant epithelial prostate cell line (RWPE-1). (A) Relative miR-205 expression levels in cell lines were assessed by quantitative reverse transcriptase-polymerase chain reaction. (B) In situ hybridization analysis of miR-205 expression in prostatic cell lines revealed that the fluorescent signals mostly were cytoplasmic in location (green) and were present only in nonmalignant RWPE-1 cells, indicating expression of miR-205 compared with cancer cell lines. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). FITC indicates fluorescein isothiocyanate.

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To examine the clinical relevance of miR-205, its expression was analyzed in carcinoma and benign prostate hyperplasia (BPH) tissue samples. Almost all carcinoma samples had significantly down-regulated miR-205 expression compared with expression in the BPH samples, and lower relative average expression was observed overall in carcinoma tissues compared with BPH tissues (Fig. 2A). An ISH analysis of miR-205 expression also was performed on tissue arrays that contained normal adjacent prostate tissue samples (n = 15) and samples of stage II (n = 14), stage III (n = 52), stage IV (n = 6), and metastatic (n = 8) carcinomas. Normal adjacent prostate tissue samples highly expressed miR-205 indicated by the BM-Purple stain. Figure 2B depicts representative ISH results indicating that normal adjacent samples expressed markedly increased miR-205 expression levels, whereas miR-205 expression was almost absent in primary and metastatic PCa samples. These ISH results suggest that expression of miR-205 is significantly high in normal tissues compared with PCa tissues.

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Figure 2. MicroRNA-205 (miR-205) is silenced in human prostate tumors. (A) Relative miR-205 RNA expression levels in benign prostatic hyperplasia (BPH) tissues (n = 24) and in tumor tissues (n = 23) were assessed by quantitative reverse transcriptase-polymerase chain reaction, and the results indicated significant expression of miR-205 in BPH samples compared with tumor samples. Vertical lines indicate average values; asterisk, P < .05 (statistically significant). (B) Photomicrographs illustrate miR-205 in situ hybridization on prostate tissue microarrays. Representative images show miR-205 expression in normal, primary, and metastatic prostate tissues (Middle); U6 staining that confirmed the preservation of intact small RNAs in the same samples (Left); and hematoxylin and eosin-stained sections that identified tumors (Right).

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MicroRNA-205 Induces Apoptosis and Cell Cycle Arrest and Impairs the Cell Viability, Migratory, Clonability, and Invasive Properties of PCa Cells

Fluorescence-activated cell sorting analysis revealed that re-expression of miR-205 led to a significant increase (10% ± 3%) in the number of cells in G0-G1—phase of the cell cycle, whereas the S-phase population decreased from 21% ± 3% to 10% ± 2%, suggesting that miR-205 caused a G0-G1 arrest in miR-205-transfected PC3 cells compared with a nonspecific miRNA control (Cont-miR) (Fig. 3A). Fluorescence-activated cell sorting analysis for apoptosis was performed using annexin-V-FITC-7-AAD dye. The percentage of total apoptotic cells (early apoptotic cells + apoptotic cells) increased significantly (11% ± 3%) in response to miR-205 transfection compared with Cont-miR (3% ± 2%), and a corresponding 8% ± 2% decrease was observed in the viable cell population (Fig. 3A). A colony formation assay revealed that overexpression of miR-205 significantly decreased the ability of metastatic PC3 cells to form colonies compared with Cont-miR-expressing cells (Fig. 3B). MicroRNA-205 caused drastic changes in cell morphology associated with growth arrest, including increased size and a broad, flattened shape 3 days (72 hours) after transfection (Fig. 3B). Cell viability after miR-205 overexpression decreased significantly (8%-30% decrease) in a time-dependent manner (from 0 hours to 72 hours) compared with Cont-miR-transfected cells (Fig. 3B), suggesting that miR-205 has an antiproliferative effect in PCa. Wound and transwell invasion assays were carried out to evaluate whether miR-205 affects the migratory and invasive capabilities of PCa cells. The average numbers of invading cells that were transfected with miR-205 were significantly lower (190 ± 11 cells) lower than the numbers of Cont-miR-transfected cells (500 ± 16 cells) (Fig. 3C). We also observed that the cells that overexpressed miR-205 cells were less proficient than the equivalent Cont-miR-transfected cells at closing the artificial wound that we created over a confluent monolayer (Fig. 3D), suggesting that miR-205 impairs the migratory capability of the metastatic PC3 PCa cells.

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Figure 3. Re-expression of microRNA-205 (miR-205) induces apoptosis, cell cycle arrest, impairs cell viability, migration, clonability, and invasive properties in PC3 cells. PC3 cells were transfected with a control microRNA (Cont-miR) or with miR-205. Untransfected and mock transfected controls were included. (A) Representative histograms and a graphic summary of cell cycle profile changes in response to miR-205 are shown. Representative quadrants and a graphic summary of apoptosis induced by miR-205 also are illustrated. (B) A colony-formation assay of PC3 cells that were transfected with miR-205 or control molecules is illustrated. The graph is representative of 3 independent experiments. The overexpression of miR-205 promoted an arrested phenotype in PC3 cells. The percentage viability of metastatic PC3 cell lines decreased in response to miR-205 in a time-dependent manner. (C) miR-205 significantly decreased the invasive behavior of PC3 cells compared with Cont-miR-transfected PC3 cells. The graph represents a summary of 3 independent experiments. (D) PC3 cells that were transfected with miR-205 exhibited less migratory behavior than Cont-miR-transfected cells.

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MicroRNA-205 Targets the IL24 and IL32 Promoters to Induce Gene Expression

Our preliminary microarray data (unpublished data) revealed that there was strong induction of the IL24 and IL32 genes in response to miR-205 transfection. To determine whether these genes have target sites for miR-205, we scanned 1-kb promoters of both genes for sequences that were complementary to miR-205 using the University of California Santa Cruz genome browser (http://genome.ucsc.edu/ accessed October 5, 2009). Our scanning analysis revealed a potential binding sequence located at positions −107 (IL24) and −610 (IL32) relative to the transcription start site in the promoters of both genes (Fig. 4A). We transfected a pre-miR-205 precursor (Ambion) and a control miR precursor (Ambion) into metastatic PC3 PCa cells and analyzed IL24 and IL32 expression 72 hours after transfection. An analysis of mRNA expression revealed a profound induction in the levels of IL24 (∼8-fold) and IL32 (∼5-fold) after miR-205 transfection compared with Cont-miR transfection (Fig. 4B). Induction of these genes was confirmed further by immunoblot analysis. Figure 4C reveals that elevated IL24 and IL32 protein levels were correlated strongly with increased mRNA expression levels. These results suggest that miR-205 induces gene expression by targeting the promoters of these genes.

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Figure 4. MicroRNA-205 (miR-205) induces expression of the interleukin (IL) tumor suppressor genes IL24 and IL32. (A) Sequences of the miR-205 target sites located at −107 base pairs (bp) (IL24) and −610 bp (IL32) are shown relative to the transcription start sites. Bases in bold face correspond to those bases in miR-205 that are complementary to the target sites, including guanine:uracil/thymine (g:u/t) wobble base-pairing (c indicates cytosine; a, adenine). (B) Relative IL24 and IL32 messenger RNA (mRNA) expression levels are illustrated in PC3 cells that were transfected with 50 nM concentrations of miR-205 or the nonspecific miRNA control (Cont-miR) for 72 hours and assessed by quantitative reverse transcriptase-polymerase chain reaction. (C) The induction of IL24 and IL32 proteins is illustrated in response to miR-205 assessed by Western blot analysis. GAPDH indicates glyceraldehyde 3-phosphate dehydrogenase.

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Sequence Specificity for Gene Induction by MicroRNA-205 and Direct Interaction Between MicroRNA-205 and Promoter Target Sequence

To determine whether the induction of IL24 and IL32 was specific to the sequence of miR-205, we synthesized 2 miR-205 mutants to create mismatches with the target sites. Mutation to 8 or 11 bases in the miR-205 sequence resulted in mutant derivatives miR-205-5MM and miR-205-3MM, respectively (Fig. 5A). Figure 5B reveals that neither miR-205-5MM nor miR-205-3MM were capable of inducing IL24 or IL32 expression, suggesting that the induction of these genes was specific to the sequence of miR-205.

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Figure 5. Sequence specificity for microRNA-205 (miR-205) is illustrated. (A) Mutations of 8 or 11 base pairs of miR-205 resulted in the mutant derivatives miR-205-5MM and miR-205-3MM, respectively. The mutated bases are shown in bold letters (u indicates uracil; c, cytosine; a, adenine; g, guanine). (B) Relative interleukin 24 (IL24) and IL32 messenger RNA (mRNA) expression levels are illustrated in PC3 cells that were transfected with 50 nM concentrations of each indicated microRNA (miRNA) duplex for 72 hours and assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cont-miR indicates a nonspecific microRNA control. Asterisks indicate P < .05 (statistically significant). (C) IL24 is a direct target of miR-205. IL24 promoter vector that contained the binding site for miR-205 was purchased from SwitchGear Genomics (Menlo Park, Calif) and cotransfected with miR-205 or Cont-miR, and luciferase activity was measured after 48 hours. Luciferase activity was normalized to total protein, and relative expression was determined for Cont-miR and miR-205. RLU indicates relative luciferase U. Asterisks indicate P < .05 (statistically significant). (D) Activation of in vitro transcription by miR-205 is illustrated. Data were derived from nuclear run-on experiments for nascent IL24 and IL32 mRNA transcription in PC3 cells transfected with miR-205 or Cont-miR duplexes, as measured by qRT-PCR and normalized to nascent glyceraldehyde 3-phosphate dehydrogenase mRNA transcription levels. Asterisks indicate P < .05 (statistically significant); RLU, relative luciferase units.

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We also performed a luciferase reporter assay to verify that a direct interaction between miR-205 and IL24 was responsible for the increased expression of IL24. The IL24 vector contained a 2.2-kb promoter sequence that included the binding site for miR-205 and an empty vector that served as a negative control. These reporter vectors were cotransfected in PC3 cells with miR-205 precursor and Cont-miR molecules that served as a negative control. The luciferase activity increased markedly after miR-205 overexpression compared with the negative control (Fig. 5C), supporting the evidence that miR-205 induces IL24 directly by targeting the promoter sequence.

MicroRNA-205 Induces In Vitro Transcription in IL24 and IL32 Genes

We performed a nuclear run-on assay to measure the relative in situ transcription rate of specific genes in intact nuclei. Within a given experiment, the nuclear run-on assay can be used to determine the level of transcription for several different genes. The isolated nuclei contain the full transcription machinery for synthesis of mRNA. Therefore, it is regarded as the gold-standard measurement of overall transcriptional activity of a specific promoter. After the transcriptional reaction, biotinylated RNA was isolated, qRT-PCR was conducted. The relative IL24 and IL32 mRNA levels reflected the transcription efficiency of both genes. Figure 5D reveals that miR-205-transfected cells had significantly higher mRNA expression levels of IL24 (7-fold) and IL32 (5-fold) compared with Cont-miR-transfected cells, suggesting that miR-205 induces the transcriptional activity of IL24 and IL32 promoters.

MicroRNA-205 Causes Enrichment of Markers for Transcriptionally Active Promoters

Several histone modifications, mainly acetylation of histones 3 and 4 and dimethylation and trimethylation of Lys 4 histone 3 (2H3K4 and 3H3K4, respectively), have been established as markers for transcriptionally active promoters. To determine whether overexpression of miR-205 results in the enrichment of active chromatin modifications in IL24 and IL32 promoters, we performed ChIP assays on miR-205-transfected and Cont-miR-transfected PC3 cells. Figure 6A,B reveals that the enrichment of almost all active chromatin modifications in response to miR-205 compared with Cont-miR was indicative of gene activation by miR-205. Therefore, the increased levels of mRNA and protein expression of IL24 and IL32 (Fig. 4B,C) in response to miR-205 overexpression were correlated with the enrichment of markers for transcriptionally active promoters in both genes.

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Figure 6. The enrichment of markers for transcriptionally active promoters by microRNA-205 (miR-205) is illustrated. (A,B) In a chromatin immunoprecipitation (ChIP) assay of PC3 cells that were transfected with either miR-205 or a nonspecific microRNA control (Cont-miR), quantification of immunoprecipitated interleukin 24 (IL24) and IL32 promoter regions was determined by quantitative real-time polymerase chain reaction (PCR) and normalized to input DNA. Signals also were confirmed by conventional PCR, and PCR products from each sample were resolved on 2% agarose gels and observed by staining with ethidium bromide. IgG indicates immunoglobulin G; H3, histone 3; H4, histone 4; 2H3K4, dimethylated Lys 4 histone 3; polymerase II. (C) The induction of downstream target genes by miR-205 is illustrated. Because miR-205 induced IL24 and IL32 expression and caused cell cycle arrest and apoptosis in PC3 cells, the expression of several downstream proteins involved in these pathways also was examined. Proapoptotic and cell cycle checkpoint proteins were induced in response to miR-205 compared with Cont-miR. BAK indicates BCL2-antagonist/killer; FLIP, flagellar biosynthesis protein; BAX, BCL2-associated X protein; p21, a cyclin-dependent kinase inhibitor; BID, BH3-interacting domain death agonist; p27, a cyclin-dependent kinase inhibitor; p38/MAPK, protein 38 mitogen-activated protein kinase; SAP-JNK, stress-activated protein-Jun N-terminal kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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Effect of MicroRNA-205 on Downstream Targets

MicroRNA-205 overexpression induced apoptosis, impaired cell migration and invasion, and caused cell cycle arrest in metastatic PC3 cells. MicroRNA-205 also induced the expression of IL24, which controls cell survival and proliferation and induces apoptosis selectively in cancer cells without harming normal cells. We examined the protein expression of various downstream genes involved in these pathways. Figure 6C reveals that the protein expression of proapoptotic genes, cell cycle regulators, and downstream targets of IL24, which are involved in the inhibition of migration, increased in PC3 cells that were transfected with miR-205 compared with Cont-miR-transfected cells (Fig. 6C). Taken together, these results suggest that miR-205 acts as tumor suppressor miRNA by directly targeting the tumor suppressor genes IL24 and IL32 through apoptotic and cell-survival pathways in PCa.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

MicroRNAs play important roles in numerous cellular processes, including development, proliferation, and apoptosis.3 Currently, it is believed that miRNAs elicit their effect by silencing the expression of target genes.15 However, in the current study, we identified 2 genes (IL24 and IL32) that have target sites complementary to miR-205 within their promoters, and the overexpression of miR-205 readily induced the expression of both genes. Several lines of evidence indicated that gene induction was specific for miR-205 and the targeted promoter sites: 1) No modifications to miR-205 or pre-miR-205 sequences were required for gene induction. We synthesized native miR-205 molecules to ensure that we were analyzing the natural miR-205 sequence function. 2) Nonspecific control molecules (Cont-miR) had no impact on target gene expression. 3) Activity required complementarity with target sequences. Mutation of the nucleotides involved in target recognition completely prevented gene induction by miR-205. 4) Selective activation of IL24 was achieved by targeting binding sites in its promoter, as confirmed in our luciferase reporter assay, and suggested that activation of this gene by miR-205 was specific for the miRNA target sites in the gene promoter. Previous reports from our laboratory and others30, 31 have revealed that small double-stranded RNAs (dsRNAs), which target the promoters of E-cadherin, p21WAF1/CIP1, vascular endothelial growth factor (VEGF), progesterone receptor, and major vault protein, readily activate gene expression. Those reports indicated an unexpected ability of small RNAs to induce gene expression.

The expression of miR-205 in cancer is controversial, because reports have indicated that it is up-regulated11, 12 and down-regulated13, 14 in tumor tissues compared with normal tissues. We observed that miR-205 was markedly down-regulated in PCa cell lines, irrespective of their androgen responsiveness, compared with normal cells. Reduction of miR-205 expression also was observed in cancer tissues compared with BPH tissues. These findings indicate that reduced miR-205 expression in PCa may be important for PCa progression. To examine this possibility, we re-expressed miR-205 in metastatic PC-3 cells. Its re-expression induced changes in cell proliferation, cell cycle progression, and cell morphology. MicroRNA-205-transfected cells had impaired clonability, migratory, and invasive capabilities. Altogether, our findings are consistent with other reports32, 33 suggesting that miR-205 acts as a tumor suppressor miRNA. A previous report33 described the tumor suppressive functions of miR-205 as involving reversal of the epithelial-to-mesenchymal transition and the down-regulation of protein kinase-C epsilon, whereas our results were distinct and identified a new function for miR-205 in inducing tumor suppressor genes by targeting their promoters.

We also observed that the IL24 and IL32 promoters were direct targets of miR-205. Luciferase activity increased significantly after miR-205 overexpression compared with negative control, indicating that miR-205 induces IL24 by directly targeting its promoter sequence. Furthermore, none of the mutated sequences of miR-205 were capable of inducing IL24 or IL32 expression, suggesting that the induction of these genes was specific to the sequence of miR-205. Our results indicate that miR-205 induced the expression of these tumor suppressor genes by targeting their promoters.

Recent studies have identified chromatin signatures that can be used to identify active promoters in human cells.34-36 Several histone modifications, mainly 2H3K4, 3H3K4, and acetylation of Lys 9/14 of histone 3, have been established as markers for transcriptionally active promoters.35, 37, 38 Because IL24 and IL32 were activated transcriptionally by miR-205, we analyzed the status of local active histone modifications, such as acetylation of histone H3 and H4, 2H3K4, and polymerase II. We observed that there was enrichment of these active histone modifications in response to miR-205 indicative of transcriptionally active promoters in the IL24 and IL32 genes, consistent with the mRNA and protein expression of both genes.

We also performed a nuclear run-on assay that measures the relative in vitro transcription rate of specific genes in intact nuclei. The isolated nuclei contain the full transcription machinery for synthesis of mRNA. The IL24 and IL32 gene transcription activity revealed by the nuclear run-on assay was increased in the nuclear extracts from cells transfected miR-205 compared with Cont-miR cells, suggesting that miR-205 induces transcriptional activity of the IL24 and IL32 promoters.

Given the functional complexity of miRNA-mediated gene regulation, it is unlikely that the effects of these molecules are limited to gene silencing. A previous report from our laboatory30 revealed that exogenously introduced dsRNAs activated the expression of E-cadherin, p21, or VEGF by targeting noncoding regulatory regions in gene promoters. Gene activation by dsRNA required the argonaute 2 protein and was associated with a loss of Lys 9 methylation on histone 3 at dsRNA target sites.30 Like these exogenously introduced dsRNAs, it is possible that miRNAs also may act to positively regulate gene expression. However, to understand the mechanism, further research will be required to identify the molecular components involved in miRNA-activated gene expression.

In summary, we have provided a proof of principle that miRNA-205 functions as a tumor suppressor in up-regulating the tumor suppressor genes IL24 and IL32 by targeting specific sites in their promoters. Enrichment of the active histone modifications in response to miR-205, indicative of transcriptionally active promoters in both genes, also was observed. The identification of this phenomenon in which cancer cells can be targeted with miRNA to turn on silenced tumor suppressor genes may have significant therapeutic potential. The use of RNA interference currently is being implemented as a gene-specific approach for molecular medicine. According to the same principle, the specific activation of tumor suppressor genes (eg, IL24, IL32) or other dysregulated genes by miRNA may contribute to a novel therapeutic approach in the treatment of PCa.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

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

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Supported by Grants RO1CA 111470 and T32DK007790 (National Institutes of Health), by a Veterans Affairs Research Enhancement Award Program, and by Merit Review grants.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES