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Keywords:

  • miRNA;
  • prostate cancer;
  • miR-34c;
  • tumor suppression;
  • proliferation

Abstract

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

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression. There have been several reports of miRNA deregulation in prostate cancer (PCa) and the biological evidence for an involvement of miRNAs in prostate tumorigenesis is increasing. In this study, we show that miR-34c is downregulated in PCa (p = 0.0005) by performing qRT-PCR on 49 TURPs from PCa patients compared to 25 from patients with benign prostatic hyperplasia. The miR-34c expression was found to inversely correlate to aggressiveness of the tumor, WHO grade, PSA levels and occurrence of metastases. Furthermore, a Kaplan–Meier analysis of patient survival based on miR-34c expression levels divided into low (< 50th percentile) and high (> 50th percentile) expression, significantly divides the patients into high risk and low risk patients (p = 0.0003, log-rank test). The phenotypic effects of miR-34c deregulation were studied in prostate cell lines, where ectopic expression of miR-34c decreased cell growth, due to both a decrease in cellular proliferation rate and an increase in apoptosis. In concordance to this, miR-34c was found to negatively regulate the oncogenes E2F3 and BCL-2, which stimulates proliferation and suppress apoptosis in PCa cells, respectively. Reversely, we could also show that blocking miR-34c in vitro increases cell growth. Further, ectopic expression of miR-34c was found to suppress migration and invasion. Our findings provide new insight into the role of miR-34c in the prostate, exhibiting tumor suppressing effects on proliferation, apoptosis and invasiveness.

MicroRNAs (miRNAs) are a class of small noncoding RNAs (19–25 nucleotides) that act as post-transcriptional regulators of gene expression. In general, miRNAs bind by imperfect base pairing to the 3′-untranslated region of target mRNA, resulting in an inhibition of translation or degradation of the mRNA.1, 2 In a breakthrough article published in 2005, Lu et al. showed that 217 human miRNAs were more accurate in determining the developmental lineage and tissue origin of tumors than ∼22,000 mRNAs.3 Over the last decade, miRNAs has also emerged as key players in tumorigenesis of several different human neoplasm's, where they can act as oncogenes or tumor suppressor genes, targeting molecules critically involved in promotion of tumor growth.

It has been shown that miRNA-34c is downregulated in a number of different malignancies, for example, neuroblastoma,4 lung cancer5, 6 and colorectal cancer.7 The miRNA-34 family is comprised of miR-34a, miR-34b and miR-34c and the family is highly conserved among different species.8 The miR-34a gene is located at chromosome 1p36, whereas the miR-34b and miR-34c genes are clustered at chromosome 11q23. Loss of heterozygosity within the 11q23 region has been detected in a number of solid cancers, including breast cancer,9 lung cancer10 and prostate adenocarcinomas (47%).11 Recently, miR-34a-c have been shown to be direct transcriptional targets of the tumor suppressor p53. In response to DNA damage, hypoxia, and oncogenic stress the transcription factor p53 has been described to activate miR-34 family genes to induce cell cycle arrest and/or apoptosis.8, 12–16

Prostate cancer (PCa) is the most common malignancy in men and the leading cause of cancer-related death for European and North American men. Recently, there have been several studies indicating that the miRNA expression profile is changed in PCa compared to benign prostatic hyperplasia (BPH).3, 17–23 The importance of miRNAs in PCa progression has further been strengthened by the fact that miRNAs have been characterized as potential oncogenes or tumor suppressors in PCa. For example, miR-449a has been proposed to act as a tumor suppressor gene by regulating cell growth and viability through the repression of histone deacetylase 1.24

In this study, we investigate the expression of miR-34a-c in PCa and BPH by qRT-PCR, and assess the biological role of miR-34c in the prostate based on a gain-of-function approach or blocking of miR-34c in vitro.

Material and Methods

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

Cell culture and tissue specimens

The cell lines were obtained from American Type Culture Collection (DU145, PC3, 22Rv1 and LNCaP clone FGC) and European Collection of Cell Cultures (PNT2 and VCaP), and cultured according to the manufacturer's recommendations. Prostatic tissues obtained by transurethral resection of the prostate (TURPs) were collected 1990–1999 in Malmö, Sweden and graded according to the WHO standard (Gleason grading was not performed routinely at this time). The material was fixed in 4% buffered paraformaldehyde and paraffin-embedded. The diagnosis was based on histopathological diagnosis in randomly selected cases with evidence of prostate adenocarcinoma in 50 patients and only BPH (i.e., no evidence of PCa) in another 25 men (Table 1). Two pathologists (one a trained uropathologist) confirmed presence of PCa and assessed the amount (%) of cancer cells using sections adjacent to those used for miRNA-analyses. One (1/50) cancer sample was found not to contain PCa in the adjacent section and was excluded from the final data set. The age range at time of TURP was 63–89, with a mean of 76 years for the men diagnosed with cancer, and 56–86, with a mean of 71 years for the men with BPH. Appropriate ethical approval has been obtained from the Ethic's Committee, Lund University and we have adhered to the Helsinki Declaration.

Table 1. Clinical characteristics of the study population
inline image

Isolation of RNA

RNA was extracted from 20-μm sections of 75 formalin fixed paraffin embedded (FFPE) prostate tissue samples. Small RNAs were extracted with a slightly modified protocol of mirVana™ miRNA Isolation Kit (Ambion, Austin, USA); the samples were deparaffinised by xylene treatment and digested by protease before the organic extraction. After washing the filter containing the small RNA, the samples were DNase treated (RecoverAll, Ambion), and washed again. From the cell lines, total RNA was isolated using Trizol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA), and treated with DNase (Promega, San Luis Obispo, USA). The RNA concentration was measured using a NanoDrop (ND-1000, Spectrophotometer, Thermo Scientific, Wilmington, DE, USA).

Reverse transcription reaction and qRT-PCR

The miRNA levels were quantified by the TaqMan MicroRNA Assays protocol (Applied Biosystems, Foster City, USA) according to the manufacturer's protocol with minor changes. Briefly, 5 or 10 ng small RNAs were reversely transcribed with miRNA specific primers. The RT product was amplified in 10-μl reactions by qRT-PCR in 384-well plates on a 7900 HT Fast Real-Time PCR System (Applied Biosystems). The samples were run in quadruplicates, and quantification was performed by the comparative Δ-Ct method. Log2-transformed values were normalized by dividing with the geometric mean of the 3 housekeeping genes RNU48, RNU66 and U47, except for the study of the expression in the cell lines where the geometric mean of RNU48, RNU66 and RNU24 was used. These 3 housekeeping genes also served as control for the RNA integrity. Along with the reverse transcription and the qRT-PCR, a no enzyme negative control and a no template control were run to exclude PCR contamination and genomic DNA.

Cell transfection

Cells were transiently transfected with either miRIDIAN microRNA Mimic (80 nM probe, Dharmacon, Lafayette, CO, USA) using Oligofectamin reagent (Invitrogen) or miRCURY LNA Knockdown probes (200 nM probe, Exiqon, Copenhagen, Denmark) using Lipofectamin 2000 reagent (Invitrogen), roughly according to the manufacturer's recommendations. Briefly, 105 cells were seeded in 6-well plates (Nunc, Roskilde, Denmark) and transiently transfected at 50–70% confluency. Control experiments were performed in parallel, transfecting cells with miRIDIAN microRNA Mimic Negative Control for overexpression (Dharmacon) and scramble-miR (Exiqon) for knockdown experiments. The miRIDIAN microRNA Mimics are small double stranded RNAs that will enter the miRNA bioprocessing pathway, but the miRIDIAN microRNA Mimic Negative Control sequence is not related to any human microRNA.

Growth assay

The Sulforhodamine B (SRB) colorimetric assay was used to estimate cell growth indirectly by staining total cellular protein content. In short, 72–120 hr after transfection, cells were fixed in 10% trichloroacetic acid and stained with 0.4% SRB (SIGMA-Aldrich, St Louis, MO) in 1% acetic acid for 15 min. Bound SRB was dissolved in 10 mM Tris base and absorbance was read at 490 nm using a microplate reader (El808, BioTek Instruments, Winooski, USA). The numbers of dead/live cells were also studied by staining cells with trypan blue.

Cell proliferation assay

The cells were incubated for 1 hr with culture media containing 5-bromo-2-deoxyuridine (BrdU, GE Healthcare, Buckinghamshire, UK) at a dilution of 1:1,000, before trypsinating and fixating the cells in ice cold 70% ethanol. The fixed cells were incubated in 2 M HCl containing 0.2 mg/ml pepsin for 20 min. After washing with PBS, the cells were incubated with 12 μl monoclonal mouse anti-BrdU antibody (Caltag laboratories, Burlingame, USA, MD5210) in blocking buffer (1% bovine serum albumin, 0.5% Tween-20 in PBS) for 1 hr at room temperature. The cells were washed in PBS and incubated with 5 μl FITC-conjugated anti-mouse IgG antibody (1 mg/ml, SIGMA-Aldrich) for 30 min, washed and stained with 20 μl propidium iodine (1 mg/ml, SIGMA-Aldrich). After incubation overnight, the cells were analyzed on a FACSort instrument (BD Bioscience, San Jose, CA).

Apoptosis/survival assay

To study apoptosis, the cells were stained with Annexin V-FITC and propidium iodine, according to the manufacturer's instructions (BD Biosciences, Franklin Lakes, USA). In brief, the cells were washed with cold PBS and trypsinized. After repeated washing, the cells were counted and 105 cells were resuspended in binding buffer (0.1 M HEPES, pH 7.4, 1.4 M NaCl, and 25 mM CaCl2). After addition of Annexin V-FITC (5 μl) and 2 μl propidium iodine (2 μg/ml), the cells were incubated in the dark for 15 min. Additional 200 μl binding buffer was added and the cells were analyzed using a FACSort instrument (BD Bioscience). The apoptotic rates of live cells are presented.

Wound healing assay

An in vitro wound healing/scratch assay was used to assess cell motility. Cells were transfected with miR-34c mimic or negative control and allowed to grow to confluence. Then a scratch was made in the cell layer with a sterile micropipette tip, the cell layer were washed twice with culture media and incubated for 3 days. The size of the scratch, from 3 independent experiments, was measured at 4 random sites in photographs taken using a Camedia C-5050 camera (Olympus Tokyo, Japan).

Invasion assay

The invasion capacity of the cells was studied using Matrigel Invasion Chambers 8.0 μm (BD Biosciences), according to manufacturer's instructions. Briefly, 2 days after transfection, the cells were seeded in serum-free medium onto the membrane of the upper chamber. Cell media, with 10% FBS was added to the lower chamber. The cells that had passed through the Matrigel coated membrane after 24 or 72 hr were fixed in paraformaldehyde and stained with crystal violet. The number of cells was counted manually in 3 independent experiments. In parallel, a negative control experiment was made where both the upper and lower chamber contained serum-free media. In addition, a SRB assay was conducted in parallel to the invasion experiment, to allow us to correct for the effect on cell growth.

Western blot

PC3 cells were transfected with miR-34c mimic or Negative Control and lysed after 96 hr with M-PER (Pierce, Rockford, USA) supplemented with Halt™ protease inhibitor cocktail EDTA-free (Pierce) and 13.4 mM EDTA. The cell suspensions were centrifuged and the supernatants collected. Protein concentration was measured and equal amount of proteins were subsequently separated on a NuPAGE 4–12% gel (Invitrogen) and transferred onto an Immobilon PVDF membrane (Millipore Corporation, Bedford, MA). The membrane was incubated with antibodies directed against E2F3 (α-E2F-3 clone PG37, mouse monoclonal IgG2a, Upstate, NY), BCL-2 (BCL-2 oncoprotein, M0887, Dako, Ely, UK) and GAPDH (α-GAPDH, MAB374, Chemicon, CA). Signals from HRP-coupled secondary antibodies (Invitrogen) were generated by ECL (ECL plus, GE Healthcare) and recorded using a CCD camera (LAS-3000, Fujifilm, Tokyo, Japan). Band intensities on Western blot were quantified and normalized to the band intensities of GAPDH, using the ImageJ software.

Statistical analysis

The median expression of the quadruplicate qRT-PCR data is presented, and analyzed by the nonparametric Mann–Whitney test, since the data are not Gaussian distributed. In samples where miR-34c expression was below the detection limit, the samples were assigned the lowest detectable level. Difference of miR-34c expression in the 4 groups BPH, WHO I, WHO II and WHO III was analyzed by Cuzick's trend test. The Kaplan–Meier method was used for survival curve analysis, and the log-rank (Mantel–Cox) test was used to determine the statistical significance between survival curves. Survival time was measured from the time of TURP and the miR-34c expression was divided into low (< 50th percentile) and high (>50th percentile). The data gained from the in vitro experiments are presented as mean ± SEM from at least 3 independent experiments and analyzed using 2-sided Student's t-test. p < 0.05 was considered statistically significant. The Graph-Pad Prism (San Diego, CA) and Statsdirect (StatsDirect, Altrincham, UK) softwares were used for statistical analysis.

Results

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

Expression of miR-34 in prostate tissue samples and cell lines

The level of miR-34c in the different prostate TURP specimens was measured using a two step miRNA specific TaqMan based qRT-PCR (median CV of the Ct values was 1.5%, with a range of 0.2–7.6%). The relative expression of miR-34c was found to be significantly lower in the PCa samples compared to the BPH samples (Fig. 1a); the median was 0.55 for the whole PCa samples cohort (3 samples were under the detection limit), and 1.1 for the BPH samples (p = 0.0005; Mann–Whitney two tailed test). We have also performed analyses for subsets containing the BPH samples compared to the PCa samples containing >10%, >20%, >30% and >50% cancer cells and they are all found to be statistically significant (p = 0.0002, p = 0.0002, p < 0.0001 and p < 0.0001, respectively). We found the miR-34c level to decrease with higher WHO grade (the median miR-34c expression in BPH = 1.1, WHO I = 0.89, WHO II = 0.71, WHO III = 0.40; Cuzick's two tailed trend test, p = 0.0002). The miR-34c levels in patients with metastases was found to be significantly lower than patients found not to have metastasis or was not investigated for metastasis since there was no suspicion at the time, p = 0.02 (Mann–Whitney two tailed test). The expression of miR-34c distinguishes aggressive from non aggressive PCa (p = 0.012, Mann–Whitney two tailed test), where the cancer was classified as aggressive if meeting either of two criteria; clinical stage T3, M1 or N1, or PCa related death. For 41 patients with PCa who had serum levels of total prostate specific antigen (tPSA) levels measured at time when the TURP was performed, we found an inverse correlation with miR-34c levels (p = 0.01; Spearman test). There was no significant correlation between miR-34c expression levels and treatment/response to treatment. The expression of miR-34c was also investigated in relation to survival time. There was a significant difference (p = 0.0018, Mann–Whitney test) in miR-34c expression for patients that died 0–3 years, compared to patients that lived for more than 3 years after TURP was performed. The same evaluation for PSA and survival time (though only data for 41 patients) did not give a significant difference. A Kaplan–Meier analysis of patient survival based on miR-34c expression levels divided into low (< 50th percentile) and high (> 50th percentile) expression is shown in Figure 1d. This threshold significantly divides the patients into high risk and low risk patients (p = 0.0003, log-rank test). We also investigated the expression of the other 2 members of the miR-34 family, but neither miR-34a nor miR-34b was significantly deregulated in our patient material (Figs. 1b and 1c). We have also found downregulation of miR-34c using the miRCURY LNA Array (Exiqon) on fresh frozen tissue (27 primary prostate tumors, 5 normal prostates) p < 0.0001, whereas miR-34a and b was not significantly deregulated (unpublished data; Ceder AY, Hagman Z, Edwards S, Missiaglia E, Clark J, Larne O and Cooper CS). The expression of miR-34c was also analyzed in 6 prostate cell lines: 22Rv1, DU145, LNCaP, PC3, VCaP and PNT2. The overall expression in the cell lines was found to be relatively low compared to that in the prostatic tissues, except for DU145 where the expression was 10 times higher compared to the other cell lines (Fig. 1e).

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Figure 1. The distribution of miR-34c (a), miR-34a (b) and miR-34b (c) in 49 patients with PCa and 25 patients with BPH determined by real time qRT-PCR. The miRNA levels are presented as the ratio of miR-34 to the geometrical mean of 3 endogenous controls (RNU48, RNU66 and U47). Kaplan–Meier analysis of patient survival based on miR-34c expression levels divided into low (< 50th percentile) and high (> 50th percentile) expression (d). Real time qRT-PCR evaluation of miR-34c levels in the prostate cancer cell lines DU145, PC3, LNCaP, 22Rv1 and VCaP, and the immortalized prostate cell line PNT2. The miRNA levels are presented as the ratio of miR-34c to the geometrical mean of 3 endogenous controls (RNU48, RNU66 and RNU24) (e).

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The effects of miR-34c on cell growth

We then investigated the phenotypic consequences of deregulation of miR-34c in prostate cells. Ectopic expression of miR-34c in different prostate cell lines resulted in a pronounced decrease in cell growth. In DU145, cells growth was decreased to 48% of control (p = 0.001) upon miR-34c transfection, and to 56% in PC3 (p = 0.001), 40% in LNCaP (p = 0.008) and 76% in PNT2 (p = 0.003), see Figure 2a. The decrease in cell growth in DU145 was confirmed by counting live cells/dead cells after trypan blue staining (74% compared to control, p = 0.02). To determine if the negative effect on cell growth was reversible, we blocked miR-34c expression with antisense LNA oligonucleotides in vitro. The growth of DU145 was 2.3-fold higher compared to control (p = 0.01) and 2.5-fold higher in PC3 (p = 0.0004) after blocking miR-34c expression, see Figure 2b.

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Figure 2. Ectopic expression of miR-34c in DU145, PC3, LNCaP and PNT2 resulted in a pronounced decrease in cell growth (a). The effect on cell growth after blocking miR-34c in DU145 and PC3 with LNA antisense probes (b). The effect on proliferation after ectopic expression of miR-34c in DU145 and PC3 (c) and the effect on apoptosis (d). Data represent mean ± SEM of 3 independent experiments.

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Ectopic expression of miR-34c suppresses cellular proliferation and promotes apoptosis

We investigated whether the reduction in cell growth upon miR-34c introduction was caused by a decrease in the cellular proliferation rate, measured by the extent of BrdU incorporation. We found that ectopic expression of miR-34c in DU145 and PC3 resulted in a considerable decrease in cellular proliferation, 50% (p = 0.0003), and 70% (p = 0.012) respectively, see Figure 2c. We also evaluated the effect on the cell cycle. Ectopic expression of miR-34c in PC3 resulted in a significant reduction of cells in S-phase (p = 0.005) with an opposite effect when blocking miR-34c expression in DU145 (p = 0.01). In parallel, we also investigated whether the decrease in cell growth could be explained by an increased rate of apoptosis. After transfecting cells with miR-34c mimic, they were stained with Annexin V-FITC and propidium iodine. There was a 4-fold increase in the apoptosis rate of PC3 cells transfected with mimic miR-34c compared to control transfected cells (p = 0.0004, Fig. 2d). There was also an increase in the apoptosis rate in DU145 cells; 150% higher compared to control (p = 0.043, Fig. 2d). A similar increase in the percentage of dead cells were also seen in DU145 with trypan blue staining (236%, p = 0.014).

Ectopic expression of miR-34c suppress migration and invasion

The effect of ectopic miR-34c expression on random migration was studied by a wound healing/scratch assay in DU145 and PC3 cells. The ectopic expression of miR-34c decreased the random migration of DU145 to 16% compared to the control (p < 0.001), and PC3 to 14% (p = 0.012), see Figures 3a and 3b. Further, we used a Matrigel invasion assay to measure the directional migration and invasion abilities of the cells after ectopically expressing miR-34c. The invasiveness of DU145 cells transfected with miR-34c mimic compared to control transfected cells was dramatically reduced (41% of control after 72 hr, p = 0.037 and 11% after 120 hr, p = 0.023) as shown in Figure 3c (data are corrected for a decrease in cell growth).

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Figure 3. Random migration by a wound healing/scratch assay. Shown are representative photographs of the scratch in DU145 cell layer 72 hr after the scratch was made (a) and a graph of the wound width in DU145 cell layer (72 hr after the scratch was made) and PC3 cell layer (96 hr after scratch was made) after ectopic expression of miR-34c (b). Matrigel invasion assay for DU145 cells ectopically expressing miR-34c at 72 and 120 hr (c). Data represent mean ± SEM of 3 independent experiments.

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miR-34c targets are involved in prostate tumorigenesis

To identify putative miR-34c targets that would mediate the phenotypic effects observed in prostate cells, we preformed in silico analyses using PicTar, Target Scan, MiRanda and miRBase. The combined list of all predicted miR-34c targets was analyzed using DAVID Bioinformatics Database functional-annotation tools (http://david.abcc.ncifcrf.gov/) to identify biological pathways that they are involved in. The top 4 KEGG pathways where the potential miR-34c targets genes are enriched were cancer related; small cell lung cancer (p = 0.001), PCa (p = 0.003), chronic myeloid leukemia (p = 0.008) and colorectal cancer (p = 0.01), and the top Biocarta pathway was “influence of Ras and Rho proteins on G1 to S transition” (p = 7.8 × 10−3). We also analyzed whether any class of protein is enriched, and found over-representation of nonreceptor serine/threonine protein kinases (p = 2 × 10−12), transcription factors (p = 5 × 10−8) and DNA helicases (p = 9 × 10−8). We investigated the effect of miR-34c on the protein levels of BCL-2 and E2F3 by Western blot, two proteins that came up in our in silico analyses and that have been seen to be regulated by miR-34c in other systems.25, 26 PC3 cells transfected with miR-34c mimic showed significantly lower levels of BCL-2; 18% (p = 0.002) and E2F3; 20% (p = 0.042) compared to control (Fig. 4).

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Figure 4. Western blot on the protein levels of BCL-2 and E2F3 after ectopically expressing miR-34c in PC3 cells. GAPDH is used as a loading control.

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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

In the present investigation the expression of miR-34c in PCa was found to be significantly lower compared to BPH, both in FFPE tissue analyzed by qRT-PCR and in array analysis of fresh frozen tissues. This indicates that miR-34c, as suggested for miRNAs in general, is not affected by FFPE treatment.27, 28 The miR-34c expression was found to inversely correlate to aggressiveness of the tumor, WHO grade, PSA levels and occurrence of metastases, indicating that miR-34c could play a part both in initiation and progression of the tumor. A lower expression of miR-34c in PCa compared to normal tissue has been detected in a previous study by Lu et al. (in their Supporting Information data).3 However, in a study by Ambs et al. miR-34c expression was shown to be higher in PCa with extraprostatic disease compared to nontumor tissue.18 The discrepancy could be due to differences in the material investigated, and the heterogenicity of the disease. The decrease of miR-34c is corroborated by the reported loss of heterozygosity at 11q23 (the locus of mir-34c) in PCa.11

There have been several studies showing that the highly conserved transcription factor p53 regulate the expression of miR-34c (and miR-34a/b).8, 12–16 Loss or mutation of p53 is rare in primary PCa but frequent in a high proportion of men with advanced PCa, and serve as an independent prognostic marker for outcome.29, 30 Lower expressions of miR-34c in malignancies have also been associated with epigenetic alterations in colorectal, breast, melanoma, lung and head/neck cancers.7, 26 Since we detect a discrepancy in the miR-34b and miR-34c expression even though they are located at the same locus, their regulation cannot fully be explained by chromosomal alterations, epigenetic silencing or p53 regulation, instead, the regulation might differ at the post-transcriptional level. Deregulation of miR-34a has been reported in a number of different malignancies,12, 31–35 and both upregulation and downregulation in PCa,17, 18 but was not found to be significantly deregulated in the PCa cohort we studied.

It has been reported that miR-34c expression increased in response to UV radiation treatment in both the androgen-dependent prostate cell line LNCaP and the androgen-independent C4-2, indicative of miR-34c not being androgen regulated.36 However, Rokhlin et al. showed that doxorubicin treatment resulted in an increased level of miR-34c in LNCaP, only upon androgen stimulation.37 The discrepancy of the two studies could be due to different methods to induce miR-34c expression through DNA damage. We found the expression of miR-34c to be very low in all prostate cell lines investigated except for the androgen-independent DU145 were expression was higher, in accordance with Rokhlin et al.37 The cell line distribution does not indicate androgen dependence, nor our study of the steady-state level of miR-34c in LNCaP cells grown in charcoal-stripped serum with/without androgen (R1881) stimulation (data not shown). There was no significant correlation between miR-34c expression levels and androgen ablation treatment or treatment failure, that is, androgen independence. The correlation between androgens, p53 and the miR-34 family, merits further investigation.

We found that miR-34c expression had a significant effect on cell growth in vitro, in all cell lines investigated, regardless of androgen dependence, p53 status or whether the cell line originated from malignant or benign cells. Similar effects on cellular proliferation rate after miR-34c overexpression has previously been described for neoplastic epithelial ovarian cells, lung carcinoma cells and gastric cancer cells.16, 25, 38 A decreased levels of E2F3 upon miR-34c overexpression is in concordance with the reduced cellular proliferation rate and less cells in S-phase, since E2F3 is involved in cell cycle regulation at the G1/S transition and stimulates proliferation in prostate cell lines.39 It has been shown that E2F3 is overexpressed in PCa and that high levels of nuclear E2F3 protein is an independent marker for poor patient survival.40 Potentially, the decrease in miR-34c expression could contribute to the increased E2F3 protein level in PCa. Our results of a reduction of cells in S-phase after ectopic expression of miR-34c are consistent with other reports on miR-34 restoration in various cancer models.8, 12, 13, 15, 16, 25, 33, 35 The effect of miR-34c on the cell cycle distribution is also in agreement with the predicted targets being enriched in the pathway of Ras and Rho proteins effecting G1 to S transition. We also detected an increased apoptosis rate after miR-34c overexpression, as has been described for colon carcinoma cells,38 and the decrease of the antiapoptotic protein BCL-2 upon miR-34c overexpression is supporting this finding. Taken together, this indicates that ectopic miR-34c expression can reduce the number of cells by a combination of a decreased cellular proliferation rate and an increased apoptosis rate in prostate cells.

In concordance with our finding that overexpressing miR-34c in vitro results in decreased migration and invasion potential, ectopic expression of miR-34c impair the ability of HT-29 colon carcinoma cells and A549 lung carcinoma cells to migrate and invade upon HGF stimulation,38 and stable combined expression of both miR-34b and miR-34c in SIHN-011B cells decreased their motility, reduced tumor growth, and metastatic nodule formation in xenograft models.26 It has also been shown that miR-34b and miR-34c cooperated to decreased anchorage-independent growth in neoplastic epithelial ovarian cells.16 This is supported by the correlation of miR-34c expression and occurrence of metastasis in PCa patients.

To summarize, our study identified a decreased expression of miR-34c in PCa, and miR-34c showed tumor suppressive abilities in vitro by affecting proliferation, apoptosis and invasion. Our results indicate that miR-34c could play a role in both the initiation of PCa and also in the progression to more aggressive and metastatic forms, suggesting that restoration of miR-34c may offer novel molecular therapy possibilities.

Acknowledgements

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

The technical assistance of Elise Nilsson at the Division of Urological Cancers at Lund University is acknowledged.

References

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

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

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