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Keywords:

  • prostate carcinoma;
  • microRNA;
  • ZNF217;
  • hnRNP-K;
  • IPO7;
  • VEGF-A;
  • miR-22;
  • miR-24;
  • miR-205;
  • miR-29b

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In primary prostate cancer (PCa), a major cause of cancer-related death in men, the expression of various microRNAs (miRNAs) is deregulated. We previously detected several miRNAs, for example, miR-24 and miR-22, as significantly downregulated in PCa (Szczyrba et al., Mol Cancer Res 2010;8:529-38). An in silico search predicted that zinc finger protein 217 (ZNF217) and importin 7 (IPO7) were potential target genes of these miRNAs. Additionally, for two genes that are deregulated in PCa (heterogeneous nuclear ribonucleoprotein K, hnRNP-K, and vascular endothelial growth factor A, VEGF-A), we identified two regulatory miRNAs, miR-205 and miR-29b. The regulation of the 3′-untranslated regions of the four genes by their respective miRNAs was confirmed by luciferase assays. As expected, the upregulation of ZNF217, hnRNP-K, VEGF-A and IPO7 could be verified at the protein level in the PCa cell lines LNCaP and DU145. ZNF217 and IPO7, which had not yet been studied in PCa, were analyzed in more detail. ZNF217 mRNA is overexpressed in primary PCa samples, and this overexpression translates to an elevated protein level. However, IPO7 was upregulated at the protein level alone. The inhibition of ZNF217 and IPO7 by siRNA resulted in reduced proliferation of the PCa cell lines. ZNF217 could thus be identified as an oncogene that is overexpressed in PCa and affects the growth of PCa cell lines, whereas the function of IPO7 remains to be elucidated in greater detail.

The induction and maintenance of tumors are facilitated by a variety of genetic changes that convert normal, resting cells into continuously proliferating cells (reviewed elsewhere1). The deregulation of microRNAs (miRNAs) has been recently recognized as one mechanism that contributes to the induction and growth of various tumors, including prostate cancer (PCa) (for a review, see Ref.2). MiRNAs are short, noncoding RNAs of approximately 19–25 nucleotides that preferentially bind to specific sequences in the 3′-untranslated region (3′UTR) of mRNAs but may also bind to the 5′UTR or the open reading frame of their targets in rare cases.3 The interaction of a miRNA and its target mRNA results in either translational repression or mRNA degradation, which ultimately leads to reduced protein synthesis. Binding to the target mRNA is accomplished via an association with the Argonaute (Ago-) proteins within the RNA-induced silencing complex (for review, see Ref.4). We have established miRNA profiles of prostate carcinoma and normal prostate tissue by a deep sequencing approach.5 Based on this analysis, we have shown that myosin VI, which is known to be upregulated in PCa, is a target for miRNAs miR-145 and miR-143, which are both downregulated in this tumor. We could subsequently identify that the Sec23A mRNA is a target for miR-200c and miR-375, which are induced in PCa, and that the Sec23A mRNA and protein are indeed downregulated in PCa. Conversely, the overexpression of Sec23A results in the reduced growth of PCa cell lines.6

In an ongoing effort to identify target genes of the miRNAs that are deregulated in PCa, we followed two bioinformatic approaches. First, we identified IPO7 and ZNF217 as potential target genes for the deregulated miRNAs miR-22 and miR-24.5 As an alternative approach, we selected two genes that are reproducibly overexpressed in PCa, vascular endothelial growth factor A (VEGF-A) and heterogeneous nuclear ribonucleoprotein K (hnRNP-K)7, 8 and found that the miRNAs miR-29b and miR-205 are predicted to target these genes. ZNF217 is located on chromosome 20q13.2, which is often amplified in different carcinomas. Overexpression of ZNF217 has been implicated in the induction of breast,9 pancreatic,10, 11 ovarian,12 cervical13 and colon carcinomas,14 as well as glioblastoma,15 possibly owing to its ability to inhibit proapoptotic signals.16 IPO7, which is located on chromosome 11, belongs to the Ran-binding protein super family.17 It is thought to act as a nuclear transport factor, and the IPO7 transcript is upregulated in colorectal carcinoma.18 VEGF-A is known to be involved in prostate carcinogenesis and as such is a target for treatment.19 hnRNP-K is a multifunctional protein involved in various aspects of RNA metabolism and is also known to be upregulated in PCa where a correlation of hnRNP-K expression and Gleason score is established.20

Herein, we demonstrate that the ZNF217, IPO7, VEGF-A and hnRNP-K genes are targets of miR-24, miR-22, miR-29b and miR-205, respectively.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Clinical samples

A total of 26 cryoconserved tumor tissue samples were available for miRNA and mRNA expression analysis. The median age of the patients at the time of diagnosis was 67.5 years (46–75 years). The Gleason score of these tumors ranged from 5 to 9. The tissue samples were macrodissected prior to RNA extraction to ensure that there was a tumor content of more than 70% in the tumor samples. The nontumor tissue, as defined by histologic examination, was prepared from the same organ as control tissue.

Cell lines, tissue cultures and antibodies

The human PCa cell lines DU145, LNCaP and human 293T were purchased from the German collection of microorganisms and cell cultures (DSMZ). Primary normal prostate fibroblasts (PNF-08) were kindly provided by Prof. Gerhard Unteregger (Department of Urology, University of Saarland Medical School). Cells were cultured as described previously.5 Anti-ZNF217 monoclonal antibodies were generated in C57/BL6 mice by immunization with a GST-ZNF217 fusion peptide. The coding sequence for amino acids 969–1049 of ZNF217 was amplified and cloned into the pGEX-4T-1 vector (GE Healthcare, Munich, Germany). The resulting GST-ZNF217 fusion protein was purified from Escherichia coli BL21/DE3 cells21 and subsequently used to immunize C57BL/6 mice according to a standard protocol.6 A clone designated ZNF 8C11 (mouse IgG2b) that reacted specifically with ZNF217 was subcloned and used for further analysis.

Plasmids

The pSG5-miR-22 expression construct was generated by polymerase chain reaction (PCR) amplification of the nucleotides 1,617,008–1,617,429 of chromosome 17 and insertion of the PCR product into the pSG5 expression plasmid (Stratagene, Heidelberg, Germany). To express hsa-miR-24, which maps on chromosome 9, the nucleotides 97,848,174–97,848,512 were PCR amplified to obtain pSG5-miR-24. To obtain pSG5-miR-205 and hsa-miR-29b, the nucleotides 209,605,356–209,605,771 of chromosome 1 and 130,562,034–130,562,416 of chromosome 7, respectively, were amplified by PCR with specific primers. The dual luciferase reporter plasmid pMIR-RL has been described elsewhere.22 The entire 3′UTR of hnRNP-K (accession number: NM_006364.2), nucleotides 1–460 of the IPO7 3′UTR (accession number: NM_006391.2), nucleotides 892–1,830 of the VEGF-A 3′UTR (accession number: NM_001171623.1) and nucleotides 1,056–1,950 of the ZNF217 3′UTR (accession number: NM_006526.2) were cloned via PCR amplification using specific primers from testis cDNA and ligated into the SpeI, SacI or NaeI restriction sites of pMIR-RL. Mutation of the predicted target site seed sequences of the pMIR constructs was carried out by site-directed mutagenesis with the QuickChange Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The primer sequences for PCR amplification and site-directed mutagenesis are listed in Supporting Information Table 1. The expression plasmid for ZNF217-HA was a kind gift from Dr. J. Torchia (London, Ontario, Canada).

Transfections, luciferase assays and Western blots

293T cells were cultivated in 24-well plates and transfected with 0.2 μg of reporter construct and 0.8 μg of miRNA expression plasmid using the Nanofectin transfection reagent (PAA, Coelbe, Germany). The luciferase assays were performed 48 hr after transfection using a Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega, Mannheim, Germany).

For Western blots, approximately 2 × 105 LNCaP or DU145 cells grown in six-well plates were transfected with 2 μg of plasmid DNA using jetPRIME (Polyplus transfection, Sélestat, France). After 48 hr, the cells were lysed with 2-fold concentrated lysis buffer (130 mM Tris/HCl, 6% SDS, 10% 3-mercapto-1,2-propandiol, 10% glycerol and 0.05% Bromophenol blue). In brief, 30 μg of the extracted proteins was separated by 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Whatman, GE Healthcare, Freiburg, Germany) by electroblotting. The primary antibodies used were anti-ZNF217 monoclonal mouse antibody 8C11, anti-hnRNP-K (D-6, Santa Cruz, Heidelberg, Germany), anti-VEGF-A (A-20, Santa Cruz), anti-IPO7 (SAB4200152, Sigma Aldrich, Munich, Germany), rabbit mAb anti-GAPDH (14C10, NEB-Cell signaling, Frankfurt, Germany) and anti-HA (3F10, Roche, Mannheim, Germany). The appropriate secondary antibodies were purchased from Sigma (Sigma Aldrich, Munich, Germany).

The extracts from primary prostate carcinoma tissue were generated using Trizol (Invitrogen, Darmstadt, Germany). Briefly, snap-frozen tissue was macrodissected to ensure that the tumor content was above 70% in the tumor samples and that cancer cells were absent in normal samples. The extraction of total RNA and protein was carried out according to the manufacturer's instructions. Twenty microgram of the protein extracts was denatured in sample buffer (62.5 mM Tris/HCL pH = 6.8, 2% SDS, 5% glycerol, 0.2 mM ethylenediaminetetraacetic acid [EDTA], 100 mM dithiothreitol, 0.05% Bromophenol blue and 0.05% Pyronin Y). The protein samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Whatman, GE Healthcare, Freiburg, Germany) by electroblotting. The membranes were incubated with the primary antibodies 8C11 and rabbit mAb anti-GAPDH (14C10, NEB-Cell signaling) and the appropriate secondary antibodies (Dianova, Hamburg, Germany). The bands were visualized by enhanced chemiluminescence (Roth, Karlsruhe, Germany) in a LAS-4000 chemiluminescence detection system (GE Healthcare).

Northern blotting

Total RNA was isolated using the peqGOLD TriFast reagent (Peqlab, Erlangen, Germany) according to the manufacturer's manual, separated by 12% denaturating urea–polyacrylamide gel and transferred to a nylon membrane Hybond N (Amersham, GE Healthcare) by semi-dry electroblotting (30 min, 15 V). The RNA was chemically crosslinked for 2 hr at 55°C and hybridized with radioactively labeled RNA probes overnight at 55°C. The antisense RNA probes were generated with the miRVana probe construction kit (Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions. After washing the membrane twice for 15 min with 5× SSC and 1% SDS and twice for 15 min with 1× SSC and 1% SDS, the membrane was exposed for at least 24 hr on a storage phosphor screen. The stripping of the nylon membrane was performed using stripping buffer (5 mM Tris, pH 8, 0.2 mM EDTA, 0.05% NaPP and 0.1% Denhardt's solution) for 2 hr at 80°C.

Quantitative real-time PCR analysis of miRNA

For miRNA analysis, 10 ng of total RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit with the miRNA-specific RT primers contained in the TaqMan MicroRNA Assays (Applied Biosystems, Darmstadt, Germany). Real-time PCR was performed with the StepOnePlus Real-Time PCR System (Applied Biosystems) using sequence-specific primers and fluorescently labeled probes for miR-22 and miR-24 (Applied Biosystems). The PCR reactions were performed in triplicate in a final volume of 10 μL containing 1× TaqMan Universal PCR Master Mix (No Amperase UNG), 1× TaqMan miRNA assay and miRNA-specific primed cDNA, corresponding to an input amount of 330 pg total RNA per real-time PCR reaction. The thermal cycling conditions were as follows: 95°C for 20 sec followed by 40 cycles of 95°C for 1 sec and 60°C for 20 sec. To quantify the miRNA expression in the tumor tissues, we used the relative quantification (ΔΔCt) method23 with RNU6b serving as an internal control. The calculated relative expression values were normalized against the RNA from PNF-08 cells. All calculations were performed with the StepOne software V 2.0 (Applied Biosystems).

Quantitative real-time PCR analysis of mRNA expression

cDNA synthesis was performed with the DyNAmo cDNA Synthesis Kit (Finnzymes Oy, Vantaa, Finland) using 200 ng of total RNA and random hexamer primers. The PCR primers for ZNF217 (fwd 5′-TTG TGT GCC TGC TGG TAG TC-3′, rev 5′-CTC TTT TGT GCC ATG CTG TTA G-3′) and for GAPDH (fwd 5′-CAT GAG AAG TAT GAC AAC AGC CT-3′, rev 5′-AGT CCT TCC ACG ATA CCA AAG T-3′) were purchased from Biomers (biomers.net, Ulm, Germany). Real-time PCRs were performed in triplicate with the StepOnePlus Real-Time PCR System (Applied Biosystems) in a total volume of 10 μL, which contained 1× TaqMan Fast SYBR Green Master Mix (Applied Biosystems), 250 nM forward primer, 100 nM reverse primer and 5 ng of cDNA with the following conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. TaqMan Assays: Sequence-specific primers and fluorescently labeled probes for IPO7 (Hs00255188_m1) and GAPDH (Hs99999905_m1) were purchased from Applied Biosystems. Real-time PCRs were performed in triplicate with the StepOnePlus Real-Time PCR System (Applied Biosystems) in a final volume of 10 μL containing 1× TaqMan Fast Universal Master Mix (Applied Biosystems), 1× Primer Assay and 2.5 ng of cDNA with the following conditions: 95°C for 20 sec followed by 40 cycles of 95°C for 1 sec and 60°C for 20 sec. mRNA expression was quantified using the ΔΔCt method23 using GAPDH as the internal control mRNA.

Cell growth and migration determination

DU145 cells (1 × 105) were seeded in six-well plates and immediately transfected using jetPRIME (Peqlab) with 110 pmol of ON-TARGETplus SMARTpool-Human ZNF217–or IPO7 or ON-TARGETplus Nontargeting Pool as a control (Dharmacon, Thermo Fisher Scientific, Karlsruhe, Germany), resulting in a final concentration of 50 nM. Cell proliferation was measured with the BrdU cell proliferation enzyme-linked immunosorbent assay kit (Roche) according to the manufacturer's instructions using an automated microplate reader (Molecular Devices, Sunnyvale, CA) The mean absorbance of control cells represented 100% cell proliferation, and mean absorbance of treated cells was related to control values to determine sensitivity. Cell proliferation (percentage of control) was determined in triplicate. The migration of cells after inhibition of ZNF217 or IPO7 (see above) was determined by a wound healing assay exactly as described previously.6

Data analysis and statistical methods

Western blots were quantified by Quantity One analysis software (Bio-Rad, München, Germany). A statistical evaluation of the luciferase assays was performed with SigmaPlot 10 (Systat, Chicago, IL) using Student's t-test statistics. The statistical analyses of the quantitative real-time (qRT)-PCR experiments (paired t-test) were performed using GraphPad Prism 4.0 (Graph Pad Software, La Jolla, CA). All statistical tests were performed as two-sided, and p-values of <0.05 were considered as significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The miRNAs miR-24, −205, −29b and −22 bind to the 3′UTR of ZNF217, hnRNP-K, VEGF-A and IPO7

We had previously shown that the levels of several miRNAs are reduced in primary cases of prostate carcinoma; for instance, miR-24 and miR-22 were downregulated more than 1.5-fold in PCa.5 We performed a bioinformatic analysis to define target genes using TargetScan (http://www.targetscan.org/). ZNF217 and IPO7 were identified as high-ranked potential targets for miR-24 and miR-22, respectively.

As an alternative approach using the Oncomine database, we selected VEGF-A and hnRNP-K as two prominent genes that are reported to be overexpressed in PCa.19, 20 An effort to find miRNAs that target VEGF-A or hnRNP-K resulted in the identification of miR-29b and miR-205. For instance, hnRNP-K is a prime target for miR-205 when it is analyzed for the potential of miR-205 to bind to its 3′UTR using the TargetScan algorithm. We found a strong downregulation of miR-205 by miRNA profiling,5 and downregulation of miR-29b in androgen-independent PCa cell lines was described by others.24 The 3′UTR regions of the target genes, including the predicted miRNA interaction sites, are schematically shown in Figure 1.

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Figure 1. Schematic representation of the reporter gene constructs and the miRNA-binding sites. For each target gene, the 3′UTR that contains the predicted miRNA interaction site is shown. Additionally, for each miRNA, the seed sequences and the mutated seed sequences in the 3′UTRs are shown. (a) ZNF217 and miR-24, (b) hnRNP-K and miR-205, (c) VEGF-A and miR-29b and (d) IPO7 and miR-22.

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To test if these miRNAs indeed exert a regulatory effect on the target genes' 3′UTR regions, we performed luciferase reporter gene analyses. The reporter gene activity of all the reporter gene constructs was significantly reduced when the respective miRNA was coexpressed in 293T cells (Fig. 2). Mutations in the binding sites of the 3′UTRs (Fig. 1) resulted in a complete loss of responsiveness toward the targeting miRNA (Fig. 2).

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Figure 2. Luciferase reporter gene analyses. The reporter gene vectors without insert (pMIR), with the 3′UTR insert or with the mutated miRNA seed sequence were cotransfected with a miRNA expression vector or a control vector in the indicated combinations. (a) ZNF217, (b) hnRNP-K, (c) VEGF-A and (d) IPO7. The reporter gene activity of the control vector experiment was set to 100% for every experiment. All values represent the mean of four independent experiments carried out in duplicate. Stars denote p < 0.05.

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The ZNF217, hnRNP-K, VEGF-A and IPO7 proteins are induced in PCa cell lines

The expression level of the four proteins in nontransformed prostate normal fibroblast (PNF-08) cells was compared to those in the LNCaP and DU145 PCa cell lines. To detect the ZNF217 protein in cell lines and tumor tissue, we developed novel mouse monoclonal antibodies. The specificity of the antibodies was determined using ectopically expressed, HA-tagged ZNF217 (Supporting Information Fig. 1). For the remaining proteins, commercially available antibodies were used. We showed an upregulation of all four proteins, ZNF217 (Fig. 3a), hnRNP-K (Fig. 3b), VEGF-A (Fig. 3c) and IPO7 (Fig. 3d) in the PCa cell lines compared to PNF-08 cells.

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Figure 3. Western blot analysis. The expression of (a) ZNF217, (b) hnRNP-K, (c) VEGF-A and (d) IPO7 was assessed in the cell lines LNCaP, DU145 and PNF-08 cells via Western blot. Staining of β-actin and GAPDH served as a loading control.

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Inhibition of ZNF217, hnRNP-K, VEGF-A and IPO7 proteins by miRNAs in the PCa cell lines

To assess the regulative capabilities of the miRNAs toward endogenous proteins, we expressed the various miRNAs in both LNCaP and DU145 cell lines and analyzed the expression levels of each protein. As expected, the miRNAs were able to downregulate their corresponding targets in either cell line (Fig. 4). We found the strongest response for miR-24, which inhibited the expression of ZNF217 by up to 80% in LNCaP and by approximately 60% in DU145 cells (Fig. 4a). The relative downregulation of the other proteins ranged from 20 to 50% in the two cell lines after ectopic expression of miR-205, miR-29b and miR-22, which is shown below the blots shown in Figures 4b, 4c and 4d. The ectopic expression of each miRNA was verified by qRT-PCR analysis (Supporting Information Fig. 2A).

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Figure 4. The regulation of protein expression by miRNAs. The PCa cell lines LNCaP and DU145 were transfected with miRNA expression vectors or a control vector. Forty-eight hours post-transfection, the protein expression of (a) ZNF217, (b) hnRNP-K, (c) VEGF-A and (d) IPO7 was determined by Western blot using the GAPDH signal as loading control.

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MiR-22 and miR-24 expression is reduced and ZNF217 and IPO7 mRNA expression is induced in the PCa cell lines

We assayed the expression levels of the four miRNAs, miR-24, miR-205, miR-29b and miR-22, in PNF-08, LNCaP and DU145 cells by Northern blotting (Supporting Information Fig. 2B). The expression of miR-22, miR-24 and miR-29b was downregulated in the PCa cell lines when compared to PNF-08 cells, whereas no expression of miR-205 was detectable by Northern blotting. We could validate these results by qRT-PCR, where we found reduced expression of miR-22 and miR-24 in DU145 and LNCaP cells compared to PNF-08 cells. The expression of IPO7 and ZNF217 mRNA was higher in both PCa cell lines than in PNF-08 cells. This inverse correlation between miRNA and their respective targets is shown in Supporting Information Figure 3A.

MiR-22 and miR-24 expression is reduced and ZNF217 and IPO7 mRNA expression is induced in primary prostate carcinoma tissues

We compared the expression of the RNAs of IPO7 and miR-22 by qRT-PCR as well as the RNA expression of ZNF217 and miR-24 in primary prostate carcinoma tissues. As shown in Figures 5a and 5b, both miR-22 and miR-24 were significantly downregulated in the tumor compared to the nontumor prostate tissue from the same organ (p = 0.0003 and <0.0001, paired t-test). We detected a significant increase in the expression of ZNF217 mRNA (p = 0.0341, paired t-test, Fig. 5b), whereas IPO7 mRNA levels were not significantly changed (Fig. 5a). In our previous profiling of the miRNA expression levels in primary tissue vs. prostate carcinoma tissue, we had found that miRNAs miR-205 was significantly downregulated in tumor tissue.5 We reanalyzed primary tissue samples by qRT-PCR and found that miR-205 was reduced by about 20-fold in tumor tissue (p < 0.0001) and that miR-29a was reduced by about fivefold (p = 0.013) (Supporting Information Fig. 3B).

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Figure 5. The correlation of miRNAs miR-24 and miR-22 and the target genes ZNF217 and IPO7 in primary PCa. (a) The correlation of miR-22 and IPO7 at the RNA level. (b) The correlation of miR-24 and ZNF217 at the RNA level. (c) Western blot analysis of ZNF217 protein expression in pairs of primary PCa and the corresponding normal tissue. (d) Western blot analysis of IPO7 protein expression in pairs of primary PCa and the corresponding normal tissue.

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ZNF217 and IPO7 protein levels are induced in primary prostate tissue

Both ZNF217 and IPO7 protein expression was measured in a representative collection of primary PCa samples and corresponding normal tissue. ZNF217 displayed a two to threefold increase in 14 out of 23 of the analyzed cases (Fig. 5c and Supporting Information Fig. 4). Similarly, the IPO7 protein level was elevated in 14 out of 23 cases (Fig. 5D and Supporting Information Fig. 4). As VEGF-A and hnRNP-K have previously been shown to be upregulated in primary PCa,20, 25 the mRNA and protein expression for these genes was not further analyzed.

Alteration of ZNF217 and IPO7 expression impacts on the growth properties of DU145 and LNCaP PCa cells

We analyzed the effect of ZNF217 and IPO7 on the growth properties of DU145 and LNCaP cells. The two cell lines were transfected with siRNAs targeting ZNF217 and IPO7, and the growth properties were assessed by counting cell numbers. An siRNA-mediated knockdown of both genes/proteins in the two cell lines resulted in a reduced growth rate, which was most pronounced at 72 hr after the transfection (Figs. 6a and 6b). Furthermore, we carried out a wound healing as described previously.6 For both ZNF217 and IPO7, we initially observed an increased wound healing at the early time points (6 and 24 hr) that was reduced, however, at the later time point (48 hr) as compared to the control (Supporting Information Fig. 5). Considering the reduced proliferation in the case of ZNF217 or IPO7 knockdown, this result points toward an increased migratory potential of PCa cells under ZNF217 or IPO7 knockdown. Taken together, our results support the notion that ZNF217 may play a role in the growth of prostate carcinoma. For IPO7, a function in tumorigenesis needs to be explored in greater detail.

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Figure 6. Cell proliferation analysis. (a) DU145 and (b) LNCaP PCa cells were transfected with siRNAs targeting ZNF217, IPO7 or a negative control siRNA. In a period of 72 hr, cell proliferation was determined by measuring BrDU incorporation. Proliferation was determined every 24 hr. The values represent a mean of triplicates.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The primary goal of our study was to identify and analyze novel potential targets of miRNAs that are deregulated in PCa. On the basis of our previous miRNA profiling, we concentrated on miRNAs miR-24 and miR-22, which were found to be downregulated in PCa.5 We reasoned that the potential targets ZNF217 and IPO7 should thus be upregulated at both the RNA and the protein levels. In addition, to uncover the miRNAs that regulate genes known to play a role in PCa, we selected and analyzed the upregulated genes hnRNP-K and VEGF-A.19, 20 We reasoned that the miRNAs that target these genes (miR-205 and miR-29b) should thus be downregulated in PCa. We discovered via luciferase assays that the miRNAs miR-24, miR-205, miR-29b and miR-22 target the 3′UTR of ZNF217, hnRNP-K, VEGF-A and IPO7, respectively. Accordingly, the ectopic expression of the miRNAs resulted in downregulation of the respective target protein in PCa cell lines.

In our previous study, using deep sequencing, we identified miR-205 as strongly downregulated in PCa tissue compared to normal prostate tissue (Ref.5, Supporting Information data). Our findings are in line with the results showing that miR-205 is significantly downregulated in PCa compared to normal tissue.26 Here, we identified miR-205 as potential regulator of hnRNP-K in silico. Our results indicate that miR-205 targets the 3′UTR of hnRNP-K and ectopic expression of miR-205 causes a 20–50% reduction in hnRNP-K protein expression in PCa cell lines. HnRNP-K as an inhibitor of androgen receptor mRNA translation regulates androgen-responsive gene expression and PCa cell proliferation.27 Barboro et al.20 showed that hnRNP-K levels within the nuclear matrix are higher in PCa compared to nontumour tissues and correlated with Gleason score and poor prognosis. A knockdown of hnRNP-K expression causes a loss of the angiogenic and migratory phenotype of prostate carcinoma cells. Furthermore, the AKT/hnRNP-K/androgen receptor (AR)/β-catenin pathway is critical for the neuroendocrine differentiation of PCa which is hypothesized to contribute to the development and growth of androgen-refractory prostate tumors (Ref.8 and references therein). In addition, it was demonstrated that miR-205 together with miR-130a and miR-203 interferes with the mitogen-activated protein kinase and AR signaling pathways in PCa.28

VEGF has been described as validated target of miR-205.29 We identified VEGF-A as new target of miR-29b in silico. This prediction was confirmed in luciferase reporter gene analysis and ectopic expression of miR-29b resulted in a 40–50% reduced VEGF-A protein expression in PCa cell lines. VEGF-A, an angiogenesis promoter, is overexpressed in PCa and BPH on the mRNA and the protein level.30 Elevated levels of VEGF are also detectable in body fluids of PCa patients.31 Noteworthy, elevated expression in the PCa tissue is significantly associated with poor differentiation, lymph node metastasis and higher pathologic stage and in addition it is an independent prognostic factor for tumor-specific survival.32 Inhibition of angiogenesis by suppression of VEGF by a decapeptide of the KISS1 protein can inhibit tumor growth in SCID mice xenografted with PC-3 PCa cells.33

We observed elevated expression of the IPO7 protein in PCa cell lines and primary cases of PCa. We could not detect a statistically significant upregulation of the IPO7 mRNA in primary prostate carcinoma tissue, which could be the result of dominant post-transcriptional regulation of the protein expression.34 C-MYC, which is one of the known proteins transported by IPO7,35 is upregulated in high-grade metastasizing PCa.36 Furthermore, IPO7 and IPO4 are involved in transport of the hypoxia-inducible factor HIF1α into the nucleus.37 Interestingly, HIF1α itself is a target of the downregulated miR-22 in colon cancer cells.38 However, the relevance of deregulated IPO7 expression in PCa remains to be established.

The major findings of this report are that ZNF217 is a target of miR-24, and its protein levels are upregulated in PCa. The amplification of this protooncogene was initially noted in a xenograft of an advanced stage PCa.39 ZNF217 is located on chromosome 20q13.2; this region is amplified in various tumors such as ovarian clear cell carcinoma,40–42 mammary carcinoma,43 head and neck squamous cell carcinoma,44 squamous cell cervical cancer13 and esophageal adenocarcinoma.45 We now extend these observations to the overexpression of ZNF217 in prostate carcinoma. We found elevated expression of ZNF217 mRNA and protein in PCa cell lines and primary PCa tissues. However, the copy number of ZNF217 genes in PCa was not investigated in our study and could additionally affect the upregulation of ZNF217. Furthermore, we showed that an alteration in ZNF217 expression has an impact on the proliferation of DU145 PCa cells. It has been shown that silencing of ZNF217 in ovarian cancer cells can reduce cell growth and invasiveness.46 A role for ZNF217 in transformation was shown by the potential of ZNF217 to immortalize primary cells.47, 48 Interestingly, ZNF217 induces the expression of the ErbB3 receptor tyrosine kinase in breast cancer cells.9 ErbB3, in turn, has been associated with the progression of PCa after androgen ablation49 and is a novel therapeutic target in the treatment of PCa.50

In summary, the downregulation of miR-24, miR-205, miR-29b and miR-22 in PCa corresponds to an upregulation of the predicted target proteins ZNF217, hnRNP-K, VEGF-A and IPO7, respectively. ZNF17 could be identified as an oncogene because it is overexpressed in PCa and affects the growth of PCa cell lines. Together with miR-24, ZNF217 and possibly IPO7 could be attractive targets for therapeutic intervention.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This research was funded by a grant of the Wilhelm-Sander-Stiftung (grant No. 2007.025.01) to B. Wullich and F. Grässer. The authors thank Ruth Nord for expert technical assistance and American Journal Experts (www.journalexperts.com) for providing language-editing service.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_27731_sm_SuppFig1.tif813KSupporting Information Figure 1.
IJC_27731_sm_SuppFig2.TIF9923KSupporting Information Figure 2.
IJC_27731_sm_SuppFig3.TIF7549KSupporting Information Figure 3.
IJC_27731_sm_SuppFig4.TIF14192KSupporting Information Figure 4.
IJC_27731_sm_SuppFig5.TIF3928KSupporting Information Figure 5.
IJC_27731_sm_SuppTab1.pdf6KSupporting Information Table 1.

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