Elevated expression of prostate cancer-associated genes is linked to down-regulation of microRNAs
© Erdmann et al.; licensee BioMed Central Ltd. 2014
Received: 1 October 2013
Accepted: 4 February 2014
Published: 11 February 2014
Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA).
In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation.
The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a.
The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.
KeywordsBiomarkers Alpha-methylacyl-CoA racemase (AMACR) Enhancer of zeste homolog 2 (EZH2) microRNAs miR-186 miR-26a Prostate cancer
Prostate cancer (PCa) is the second most frequent tumor and the sixth leading cause of cancer-related death among males worldwide . Even though early detection of PCa has dramatically increased since the introduction of serum prostate-specific antigen (PSA) measurement, the lack of specificity of PSA as a tumor marker results in a high rate of unnecessary biopsies . Consequently, various attempts have been made to identify new biomarkers that allow the detection of PCa at an early stage as well as the discrimination between benign and malignant alterations of the prostate. In previous studies, we have analyzed selected transcript markers such as AMACR, EZH2, PSGR, PSMA and TRPM8 among others in PCa tissue specimens. All of these markers were significantly up-regulated in PCa tissue compared to non-malignant prostate tissue and thus, could be of clinical importance for diagnostic purposes [3–6].
Originally identified as an enzyme that is involved in the metabolism of fatty acids AMACR (alpha-methylacyl-CoA racemase) is also highly over-expressed in PCa and its immunohistochemical detection is currently used by pathologists to achieve definitive diagnosis of PCa [7, 8]. It has been shown that AMACR can modify the growth of PCa cells in an androgen-independent manner . EZH2 (enhancer of zeste homolog 2) is a member of the polycomb-group family and functions as a transcriptional repressor . As an oncogene it is frequently up-regulated in hormone-refractory metastatic PCa suggesting a critical role for EZH2 in disease progression . PSGR (prostate-specific G-protein coupled receptor; synonym: olfactory receptor, family 51, subfamily E, member 2 (OR51E2)) is a member of the G-protein-coupled olfactory receptor family that is predominantly expressed in the human prostate [12, 13]. PSGR has been described to be over-expressed in PCa tissue [13, 14] and a multiplexed model based on the detection of PSGR and PCA3 (prostate cancer gene 3) in urine improved the specificity for PCa prediction . PSMA (prostate-specific membrane antigen; synonym: folate hydrolase 1 (FOLH1)) is a cell-surface antigen with abundant and virtually universal expression in PCa which increases as the cancer progresses [16, 17]. Since PSMA is an antigen that is highly specific for PCa tissue its targeting can be used for in vivo imaging and immunotherapy of PCa [18, 19]. TRPM8 (transient receptor potential cation channel, subfamily M, member 8; synonym: Trp-p8) is involved in the regulation of the intracellular Ca2+ concentration and exhibits an elevated expression in PCa [20, 21]. TRPM8 is an androgen-responsive gene and essential for the survival of PCa cells .
The tumor-specific up-regulation of the aforementioned genes suggests a functional role for these genes in the development and progression of PCa. However, the genetic and epigenetic mechanisms that lead to their up-regulation are mainly unknown. The demonstrated abnormal expression patterns could be associated with a deregulation of microRNA (miRNA) expression. MiRNAs are small (~22 nucleotides) non-coding RNAs that are involved in a variety of oncogenic pathways . As post-transcriptional regulators they bind to the 3′-untranslated region (3′UTR) of their target mRNA resulting in either translational repression or mRNA degradation [23, 24]. Depending on their target genes miRNAs can either function as oncogenes or tumor-suppressors .
It has been reported that miRNAs have distinct expression profiles in various human cancers [25–27]. Several profiling studies have also shown that the expression of miRNAs is commonly altered in PCa compared to normal tissues [25, 28–33]. A deregulation of the miRNA expression consequently leads to an altered interaction with their respective mRNA targets and thus, promotes abnormal cellular functions [34, 35]. To evaluate the influence of miRNAs on the onset or progression of PCa it is therefore of utmost importance to identify and analyze potential interactions between PCa-associated genes and their putative miRNA regulators. However, only few studies have assessed such a connection between a miRNA deregulation and an up-regulation of PCa-specific genes. Of the PCa-associated genes investigated in this study a miRNA-mediated regulation has been reported only for EZH2 so far [36–40].
The aim of this study was to identify miRNAs that could potentially regulate the expression of genes that are known to be up-regulated in PCa. Subsequently, the expression levels of both the candidate miRNAs and the PCa biomarkers were analyzed in malignant and non-malignant prostate tissues. Furthermore, the miRNA expression data were evaluated with regard to a potential correlation with the expression levels of the PCa-associated genes as well as with clinicopathological parameters. In an initial assessment the influence of exogenously administered miR-26a on the mRNA and protein expression of its known target EZH2 as well as its potential new target gene AMACR was investigated in various PCa cell lines. Subsequently, target validation for miR-26a was conducted by a luciferase reporter assay.
In silicomiRNA prediction
To identify miRNAs that might target the PCa-associated genes AMACR, EZH2, PSGR, PSMA, and TRPM8 the following publicly available bioinformatic prediction programs as well as a database of experimentally supported miRNA targets were used: TargetScanHuman v5.1, TargetScanS, PicTar (based on conservation in mammals), MicroCosm Targets, microRNA.org (release 03/2009), Human miRNA Targets (optimized intersection: PicTar, TargetScanS), DIANA microT v3.0 and DIANA TarBase v5.0 (Additional file 1: Table S1). For subsequent analyses miRNAs were considered that were predicted (i) by multiple algorithms per gene or (ii) for more than one gene.
Clinicopathological data of the patients
Total patient number
Age at surgery [years]
65 (49 – 78)
72 (50 – 86)
Pre-operative PSA [ng/ml]
10.2 (2.8 – 113.0)
2.6 (0.2 – 46.2)
pT3 + 4 (nonorgan-confined)
< 7 (low)
> 7 (high)
Lymph node status
Distant metastases at prostatectomy
The human PCa cell lines DU-145 (HTB-81), PC-3 (CRL-1435) and LNCap (CRL-1740) were obtained from the American Type Culture Collection (ATCC) and maintained at standard conditions (37°C, humidified atmosphere containing 5% CO2) without antibiotics. DU-145 and PC-3 cells were cultured in DMEM (4.5 g/l glucose) supplemented with 10% fetal bovine serum (FBS), 1% 1 M HEPES buffer and 1% MEM non-essential amino acids, whereas LNCap cells were grown in RPMI-1640 including 10% FBS and 1% MEM non-essential amino acids (all from Life Technologies).
MiRNA mimics, siRNAs and transient transfection
The mimic for miR-26a (PM10249) and the Pre-miR Negative Control #1 (miR-CON) were obtained from Life Technologies. Specific small interfering RNAs (siRNAs) directed against AMACR (siR-AMACR; sense: GAGAUUUAUCAGCUUAACU, antisense: AGUUAAGCUGAUAAAUCUC) and EZH2 (siR-EZH2; sense: CACAAGUCAUCCCAUUAAA, antisense: UUUAAUGGGAUGACUUGUG) as well as the negative control siRNA (siR-CON; SR-CL000-005) were synthesized by Eurogentec. Cells were washed with PBS and transfected for 4 h in serum-free OptiMEM (Life Technologies) using DOTAP liposomal transfection reagent (Roche) according to the manufacturer’s instructions. The final concentrations of the transfectants and their respective controls were either 100 nM (miRNA mimic) or 150 nM (siRNAs). After 4 h, transfection medium was replaced by fresh cell culture medium and cells were incubated for another 48 h. For further analyses cells were then harvested by trypsin/EDTA treatment.
RNA isolation and cDNA synthesis
RNA was isolated from cells using peqGOLD TriFast (Peqlab) and from tissue cryosections either using Invisorb Spin Tissue RNA Mini Kit (Invitek; for subsequent mRNA analysis) or peqGOLD TriFast (for subsequent miRNA analysis) according to the manufacturers’ recommendations. For mRNA analysis in tissues and cells, reverse transcription of 500 ng RNA into cDNA was carried out using SuperScript II Reverse Transcriptase (Life Technologies) and random hexamer primers (GE Healthcare) according to the manufacturers’ recommendations. For miRNA analysis in tissue samples, a total of 400 ng RNA was reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit and Megaplex RT Primers (Human Pool A; both Life Technologies) which allows for reverse transcription of up to 381 miRNAs in a single reaction.
Quantitative polymerase chain reaction (qPCR)
Gene expression of AMACR, EZH2, PSGR, PSMA and TRPM8 as well as of the reference gene TBP (TATA box binding protein) was measured by qPCR using the LightCycler FastStart DNA Master Hybridization Probes Kit and the LightCycler 1.5 instrument (both Roche). Primers and probes are listed in Additional file 1: Table S2; qPCR conditions are summarized in Additional file 1: Table S3. The mRNA copy number of a single marker was calculated in relation to the amplification product amounts of external standards as described previously . All qPCR measurements were carried out at least twice as independent PCR runs for each cDNA sample. Samples were measured for a third time if differences of >30% occurred. The means of all measurements were used for further calculations. Relative expression levels of PCa related markers were obtained by normalization to the reference gene TBP.
The expression of the selected miRNAs was quantified by miRNA-specific TaqMan MicroRNA Assays (Life Technologies) according to the manufacturer’s instructions using the TaqMan Gene Expression Master Mix and the LightCycler 480 instrument (both Roche) (Additional file 1: Table S3). The following TaqMan MicroRNA Assays were used: 002253 (hsa-miR-101), 002284 (hsa-miR-138), 002285 (hsa-miR-186), 002099 (hsa-miR-224), 000405 (hsa-miR-26a), 000407 (hsa-miR-26b), 000563 (hsa-miR-374a), 001274 (hsa-miR-410), 001515 (hsa-miR-660) and 001006 for the reference RNA (RNU48). RNU48 was selected for normalization purposes due to its reported biological stability and its usefulness as a reference molecule for miRNA expression analyses in PCa and other cancer tissues [41–43]. Automatic second derivative analysis was applied for the determination of the crossing points (CP). Each CP was determined twice in independent qPCR runs and the mean value was used for further calculations. If the mean deviation of both CP values exceeded 0.25, a third measurement was done and included in the calculation of the mean. Standard curves were used to determine the copy number of a single miRNA. Relative expression levels of the miRNAs were obtained by normalization to the reference RNA RNU48.
In tissue samples the fold expressions of PCa-associated genes as well as of miRNAs were determined relative to the median relative expression in Tf or BPH tissues. For transfection experiments the fold expressions were calculated using the ΔΔCP method.
Heat map generation
Heat map generation was carried out using the Genesis software package. Relative expression data were log-transformed and fully normalized for genes and miRNAs.
Western Blot analysis
Protein separation and subsequent Western blotting were performed as described previously . Membranes were probed with primary antibodies against AMACR (1:1000; Cell Signaling, clone 2A10), EZH2 (1:750; Cell Signaling, clone AC22) and α-tubulin (1:5000; Calbiochem, clone DM1A); the latter served as a loading control. The secondary polyclonal rabbit anti-mouse immunoglobulin HRP-linked antibody (1:1000; Dako, P0260) as well as the Enhanced Chemiluminescence Kit (GE Healthcare) were used for visualization. Quantification of the protein content was performed by means of computer-assisted videodensitometry (Quantity One Basic, Bio-Rad).
Construction of plasmid vectors and luciferase reporter assay
A putative binding site of miR-26a within the 3′UTR of AMACR was identified using the target prediction tool of microRNA.org (Additional file 1: Table S1). To construct luciferase reporter vectors, oligonucleotides (Biomers) comprising the wildtype or mutated binding site were inserted downstream of the Firefly luciferase gene into the pmir-GLO Dual-Luciferase miRNA Target Expression Vector (Promega) according to the manufacturer’s instructions. The insert regions in the vectors were sequenced (GATC Biotech) to verify incorporation of the respective target sequence. The resulting vectors are referred to as pmir-GLO-A26a (AMACR-specific miRNA-26a-binding sequence; AAC ACA CTG AGG AGA TAC TTG AA) and pmir-GLO-Amut26a (mutated AMACR-specific miRNA-26a-binding sequence; AAC ACA CTG AGG CGA GAC CCA AA). Nucleotides in bold indicate changes introduced within the target sequence to generate the mutant form.
For luciferase reporter assays, DU-145 cells were cultured in 24-well plates and co-transfected with 1.5 μg of the indicated vector and 100 nM of miR-26a mimic or miR-CON using Lipofectamine 2000 (final concentration 20 ng/μl; Life Technologies) for 24 h. Following incubation with fresh cell culture medium for another 24 h, cells were lysed and analyzed for luciferase activity using the Dual-Glo Luciferase Assay System (Promega) and a Mithras LB 940 Multimode Microplate Reader (Berthold) according to the manufacturers’ instructions. Following background adjustment, Firefly luciferase activity was normalized to Renilla luciferase activity. The normalized luciferase activity was then compared to that of the pmir-GLO-A26a vector co-transfected with miR-CON. For each transfection, luciferase activity was averaged from three replicates.
Statistical analyses were carried out with the PASW Statistics 18.0.0 (SPSS) software. Correlations were assessed by Spearman’s rank correlation coefficients. Group comparisons were conducted as indicated. A p value <0.05 was defined to be statistically significant; p < 0.1 was considered as a statistical trend.
Up-regulation of PCa-associated genes
Differentially expressed genes between malignant and non-malignant prostate tissues samples
Median relative transcript levels
Median fold expressions
Tu vs Tf[median]
Tu vs BPH[median]
(n = 50)
(n = 50)
(n = 30)
Identification of putative miRNA regulators for PCa-associated genes
Results of the in silico analyses and the Spearman rank correlation
Down-regulation of selected miRNAs in PCa tissues
Differentially expressed miRNAs between malignant and non-malignant prostate tissues samples
Median relative transcript levels (x10-3)
Median fold expressions
Tu vs Tf[median]
Tu vs BPH[median]
(n = 50)
(n = 46)
(n = 30)
Expression levels of PCa-associated genes and selected miRNAs depending on clinicopathological parameters
Furthermore, the relative transcript levels of the relevant genes as well as of the selected miRNAs were compared with regard to the different clinicopathological parameters. With the exception of TRPM8 none of the relevant genes showed a significant association with age, serum PSA concentration, tumor stage, Gleason score or initial metastases at prostatectomy (Additional file 1: Table S5). Solely the expression of TRPM8 was with 25.14 significantly lower in patients with nonorgan-confined tumors (pT3 + 4, n = 27) compared to organ-confined tumors (pT2, n = 23) with 52.44 (p = 0.03) (Additional file 1: Table S5).
Correlation between the expression of PCa-associated genes and their putative miRNA regulators
A possible correlation between the expression of the selected miRNAs and of their putative target genes was analyzed by Spearman rank correlation using the expression data gained from all tissue specimens (50 Tu, 46 Tf, 30 BPH). The expression levels of specific miRNAs showed weak to moderate inverse correlations with the expression levels of their putative target genes. The Spearman correlation coefficients (rs) ranged from -0.107 to -0.551 (Table 3). Except for miR-101 and miR-26b these correlations were statistically significant. However, a statistical trend was found for the combinations miR-101/EZH2 (rs = -0.156, p = 0.081) and miR-26b/AMACR (rs = -0.154, p = 0.086). Overall, the strongest correlations with the expression of their putative target genes were observed for miR-186, miR-26a and miR-224 (Table 3).
Effects of exogenous miR-26a on the expression of selected target genes in PCa cell lines
Transcript expression of miR-26a in PCa cell lines
Median relative transcript levels (x10-3)
35.1 ± 12.3
44.0 ± 29.8
66.7 ± 37.1
miR-CON (100 nM)
36.6 ± 14.4
33.2 ± 23.1
52.2 ± 36.3
miR-26a (100 nM)
30895.2 ± 13836.0α,β
16047.6 ± 13441.3α,β
11042.1 ± 6940.7α,β
Following exogenous administration of the miR-26a mimic a significant increase of this miRNA was observed in all three cell lines (Table 5). An over-expression of miR-26a diminished the AMACR transcript and protein level by about 20-60% and 20-50%, respectively, depending on the cell line (Figure 3A, Figure 4A, C). In contrast, treatment with the mimic for miR-26a did not produce a distinct inhibition of EZH2 mRNA and protein expression in any cell line (Figure 3B and 4B).
Direct regulation of AMACR by miR-26a
It is widely accepted that oncogenesis and tumor progression is initiated through a deregulated expression of certain genes which then leads to the malignant transformation of the affected cells. In previous studies we have shown that genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 are tumor-specifically up-regulated in PCa compared to benign tissue and thus, could be used for diagnostic purposes [3–6], whereupon the observed PCa-specific up-regulation of the relevant genes was comparable to those in the present study. Consequently, the elevated expression of the aforementioned genes can deeply impact the growth and survival of PCa cells eventually resulting in oncogenesis and/or tumor progression [9, 11, 13, 22, 45]. Therefore, such PCa-associated genes could represent suitable diagnostic tools as well as promising targets for the therapeutic intervention. However, the identification and characterization of the underlying mechanisms for the deregulation of these molecular markers are crucial for the understanding of the biology and clinical course of the disease. The demonstrated abnormal expression patterns could be associated with a deregulation of miRNAs which serve as post-transcriptional regulators of their target gene expression [23, 24]. In accordance, miRNAs are aberrantly expressed in several types of cancers [25–27] and thus, could influence oncogenesis and tumor progression via an altered miRNA-target gene-interaction. Using in silico analyses nine miRNAs (miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660), which could potentially regulate the expression of the PCa-associated genes, have been selected for further analysis.
By using a qPCR approach it was revealed that the selected miRNAs are down-regulated in PCa compared to matched non-malignant tissue or BPH, respectively. Several studies have also demonstrated distinct miRNA expression profiles for PCa compared to normal prostate tissue [25, 28–33]. However, a comparison of the studies by Ambs et al., Volinia et al. and Porkka et al. showed that there are no overlapping subsets between the differentially expressed miRNAs analyzed in these studies . The observed inconsistencies can mainly be attributed to different methods of tissue collection, RNA isolation and miRNA detection . Therefore, a consensus on PCa-specific miRNA alterations has not been established to date.
The above mentioned discrepancies have also been observed for some of the miRNAs evaluated in this study. To begin with, some studies demonstrated a down-regulation for miR-101 , miR-224 , miR-26a , miR-26b  and miR-410  in primary PCa samples compared to normal prostate tissue which is consistent with our results. In contrast to our data and to some of the aforementioned profiling studies, up-regulated expression levels in PCa tissues have been demonstrated for miR-101 , miR-26a [25, 28] and miR-26b . However, the results of the cited profiling studies were obtained by microarray or deep sequencing analysis and have not been validated by qPCR with the only exception of miR-26a which was confirmed to be up-regulated in a small subset of 10 prostatic tumors .
In agreement with our results and also based on an assessment by qPCR, a significant down-regulation in primary PCa compared to benign samples was noted for miR-101 , miR-26a  and miR-224 , whereas miR-26b was only diminished by trend . In a small sample cohort, miR-138 was up-regulated in high grade tumors (Gleason score ≥8; n = 14) versus normal epithelium (n = 10), which is contradictory to our results . Upon reviewing the current literature miR-186, miR-374a and miR-660 have not been demonstrated to be differentially expressed in primary PCa compared to benign prostate tissue to date. Therefore, this is the first study reporting that miR-186, miR-374a and miR-660 are significantly down-regulated in primary PCa compared with benign samples.
Furthermore, none of the profiling studies evaluated associations of the particular miRNAs with clinicopathological parameters or has further analyzed them with regard to the regulation of potential target genes [25, 28–32]. Only in the qPCR-based study by Mavridis et al., miR-224 expression was reported to be gradually decreased as Gleason score and tumor stage progressed and also to be associated with a favorable prognosis . In the present study, the miR-224 transcript levels were also frequently lower albeit not significantly in tumors that were more aggressive or in an advanced disease stage. Eventually, a significant association with clinicopathological features was only observed for miR-186. A decreased miR-186 expression was significantly linked to more aggressive and advanced tumors indicating that down-regulation of miR-186 in PCa could be a factor of disease progression.
The present study also demonstrated that the deregulation of the miRNAs is linked to an increase of the transcript levels of their putative target genes. Except for miR-101 and miR-26b the expression levels of the evaluated miRNAs showed significantly weak to moderate inverse correlations with the expression levels of their putative target genes. Among the miRNAs included in this study miR-101 [36, 40], miR-138 [37, 39], miR-26a [36, 38] and miR-26b  are of particular interest as some studies have already identified EZH2 as one of their direct target genes. This was also reflected here by the high prediction rate of these miRNAs for EZH2 in the in silico analyses. The link between these miRNAs and EZH2 has been demonstrated in numerous experimental settings amongst others investigating the importance of this regulatory mechanism for the onset and progression of various types of cancer including PCa. In various PCa cell lines, over-expression of miR-101, miR-26a and miR-26b could lead to repression of both EZH2 mRNA and protein as well as to a reduced cellular proliferation suggesting a tumor-suppressive function for these miRNAs in PCa [36, 38, 40].
For some initial continuative analysis, we focused on miR-26a as this miRNA has already been identified as a direct regulator of EZH2 in PCa [36, 38]. Moreover, the down-regulated expression of miR-26a in clinical PCa samples has been shown to be significantly inversely correlated with EZH2 levels with a Spearman correlation coefficient of -0.516 (p = 0.0013) . In the present study, there was also a significant inverse correlation between the expression of EZH2 and miR-26a (rs = -0.383, p < 0.01). The differences between the two studies might be partly explained by the use of different sample cohorts. Koh et al. analyzed the expression of miR-26a and EZH2 in 36 prostate samples (18 Tu, 18 Tf) , whereas we conducted the expression analyses in a larger cohort of 126 prostate tissue samples (50 Tu, 46 Tf, 30 BPH) and thus, may have gained a higher statistical reliability. However, in the present study, miR-26a failed to decrease EZH2 when administered exogenously to PCa cells. This finding is similar to the results reported by the study of Cao et al. in which miR-26a reduced EZH2 protein levels only in DU-145 cells . In contrast, Koh et al. reported that over-expression of miR-26a repressed both EZH2 mRNA and protein in DU-145, PC-3 and LNCap cells . The authors attributed this discrepancy to methodical differences which cannot be excluded.
In addition to EZH2, AMACR was also identified as a target gene possibly regulated by miR-26a. The putative link between AMACR and miR-26a was reflected by a moderate inverse correlation of their expression levels in prostate tissue (rs = -0.335, p < 0.01). In vitro results gathered in this study demonstrated that miR-26a can potently repress the mRNA and protein expression of AMACR depending on the cell line. A direct regulatory effect of miR-26a on the newly identified target gene AMACR was confirmed by luciferase reporter assay. Co-transfection of DU-145 cells with the miR-26a mimic and the luciferase reporter vector containing the wildtype AMACR binding site produced a decrease in luciferase activity by about 40%, whereas co-transfection with the luciferase reporter vector containing the mutated AMACR binding site did not lead to a reduction in luciferase activity. Taken together, this is the first study showing that the expression of AMACR can directly be regulated by a miRNA.
Overall, this is the first study that demonstrated potential interactions between the PCa-associated genes AMACR, PSGR, PSMA and TRPM8 and specific miRNAs. Notably, strong correlations were also observed between miR-186 and its putative target genes AMACR and PSMA as well as between miR-224 and its proposed target gene AMACR. Further research is warranted to confirm a direct regulatory effect of these miRNAs on their potential target genes.
In conclusion, this study demonstrated that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction. A preliminary in vitro assessment showed that exogenous administration of miR-26a resulted in a decreased expression of AMACR mRNA and protein depending on the cell line. By using a luciferase reporter assay, AMACR was confirmed as a direct target of miR-26a.
Benign prostatic hyperplasia
Enhancer of zeste homolog 2
Fetal bovine serum
Prostate cancer gene 3
Prostate-specific G-protein coupled receptor
Prostate-specific membrane antigen
Quantitative polymerase chain reaction
small interfering RNA
TATA box binding protein
Transient receptor potential cation channel, subfamily M, member 8
This work was supported by the Wilhelm Sander-Foundation (Grant number: 2010.041.1). We acknowledge support by the German Research Foundation and the Open Access Publication Funds of the TU Dresden. The funding body did not have any role in the design of the study, collection, analysis, and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.
The authors are grateful to Jana Scholze, Andrea Lohse-Fischer, Ulrike Lotzkat and Silke Tomasetti for their excellent technical assistance. The authors would like to express their gratitude to Prof. Rainer Koch and to Dr. Alexander Herr for their support with the statistical analyses and the heat map generation, respectively.
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