Elevated expression of prostate cancer-associated genes is linked to down-regulation of microRNAs

Abstract

Background: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Figures

Figure 1

Relative transcript levels of miR-186 in PCa samples with regard to clinicopathological parameters. (A) Comparison of the miR-186 expression levels in organ-confined (pT2, n = 23) versus nonorgan-confined tumors (pT3 + 4, n = 27). (B) Comparison of the miR-186 expression levels in patient samples without any initial metastases (N0M0, n = 40) versus those with initial metastases (N+/M+, n = 10). Transcript levels of miR-186 were normalized to RNU48. P values were calculated by the Mann–Whitney U test with a two-sided 95% confidence interval. Outliers are indicated by circles; extreme values are indicated by asterisks.

Figure 2

Matched miRNA and target gene expression with regard to the prostate tissue specimens. Depicted are exemplary scatter plots with trend lines for (A) miR-186/AMACR, (B) miR-186/PSMA, (C) miR-26a/TRPM8 and (D) miR-26a/EZH2. Transcript levels of miRNAs and target genes were normalized to RNU48 and TBP, respectively.

Figure 3

Effect of miR-26a mimic and siRNAs on target gene mRNA expression in PCa cell lines. Transcript levels of (A) AMACR and (B) EZH2 were determined by qPCR and normalized to TBP. Normalized values are shown relative to the corresponding control treatments (100%): miR-CON (100 nM) for treatment with miR-26a mimic and siR-CON (150 nM) for treatment with siRNAs, respectively. Values represent averages of two to five independent experiments with their mean deviation. A one-tailed paired t-test was used to compare differences between cells treated with miR-26a mimic or siRNAs and the respective control treated cells (miR-CON or siR-CON): *p < 0.05, **p < 0.01.

Figure 4

Effect of miR-26a mimic and siRNAs on target gene protein expression in PCa cell lines. Protein levels of (A) AMACR and (B) EZH2 were determined by Western Blot and normalized to α-tubulin. Normalized values are shown relative to the corresponding control treatments (100%): miR-CON (100 nM) for treatment with miR-26a mimic and siR-CON (150 nM) for treatment with siRNAs, respectively. Values represent averages of two to five independent experiments with their mean deviation. A one-tailed paired t-test was used to compare differences between cells treated with miR-26a mimic or siRNAs and the respective control treated cells (miR-CON or siR-CON): *p < 0.05, **p < 0.01, #p < 0.1 (statistical trend). (C) Exemplary Western Blots for the detection of AMACR protein following treatment with 100 nM miR-26a mimic or miR-CON are depicted. Alpha-tubulin served as loading control. Lines indicate that the sample lanes were not adjacent in the original gel. However, samples per cell line were retrieved from the same experiment and each sample was simultaneously probed for AMACR and α-tubulin.

Figure 5

miR-26a directly targets AMACR. (A) The mature miR-26a sequence aligned with the predicted binding site within the 3′UTR of AMACR is depicted. (B) Luciferase activity was measured in DU-145 cells co-transfected with 100 nM miR-26a mimic and 1.5 μg of the wildtype (pmir-GLO-A26a) or mutated (pmir-GLO-Amut26a) luciferase reporter vector for AMACR. Normalized values are shown relative to the control treatment with 100 nM miR-CON and the wildtype luciferase reporter vector (100%). Values represent averages of six independent experiments with their mean deviation. A one-tailed paired t-test was used to compare differences between the respective treatments and the control treated cells (pmir-GLO-A26a + miR-CON): *p < 0.05.