miR-21: an oncomir on strike in prostate cancer

Abstract

Background: Aberrant expression of microRNAs, small non-coding RNA molecules that post-transcriptionally repress gene expression, seems to be causatively linked to the pathogenesis of cancer. In this context, miR-21 was found to be overexpressed in different human cancers (e.g. glioblastoma, breast cancer). In addition, it is thought to be endowed with oncogenic properties due to its ability to negatively modulate the expression of tumor-suppressor genes (e.g. PTEN) and to cause the reversion of malignant phenotype when knocked- down in several tumor models. On the basis of these findings, miR-21 has been proposed as a widely exploitable cancer-related target. However, scanty information is available concerning the relevance of miR-21 for prostate cancer. In the present study, we investigated the role of miR-21 and its potential as a therapeutic target in two prostate cancer cell lines, characterized by different miR-21 expression levels and PTEN gene status.

Results: We provide evidence that miR-21 knockdown in prostate cancer cells is not sufficient per se i) to affect the proliferative and invasive potential or the chemo- and radiosensitivity profiles or ii) to modulate the expression of the tumor-suppressors PTEN and Pdcd4, which in other tumor types were found to be regulated by miR-21. We also show that miR-21 is not differently expressed in carcinomas and matched normal tissues obtained from 36 untreated prostate cancer patients subjected to radical prostatectomy.

Conclusions: Overall, our data suggest that miR-21 is not a central player in the onset of prostate cancer and that its single hitting is not a valuable therapeutic strategy in the disease. This supports the notion that the oncogenic properties of miR-21 could be cell and tissue dependent and that the potential role of a given miRNA as a therapeutic target should be contextualized with respect to the disease.

Figure 1

Characterization of PCa cells for miR-21 and PTEN expression. (A) RT-PCR and western immunoblotting, and (B) RNase protection assay showing the basal expression levels of PTEN and miR-21 in DU145 and PC-3 cell lines. (C) qRT-PCR showing the down-modulation of free miR-21 in DU145 (grey bars) and PC-3 cells (white bars) exposed to LNA21. Data are reported as relative quantity (RQ) of LNA21- over LNAScr-treated cells and represent mean values ± SD of at least three independent determinations.

Figure 2

Analysis of the effects of miR-21 knockdown on PCa cell behavior. (A) Growth curves of untreated (white triangle), LNAScr (black circle)- or LNA21 (white circle)- transfected DU145 and PC-3 cells. (B) Analysis of the migrating (black bars) and invading (white bars) capabilities of PCa cells (number of cells/mm²) at day 3, in untreated (UNT) and LNAScr- or LNA21-transfected cells. (C) Time-course determination of caspase-3 catalytic activity in untreated (black bars) and LNAScr- (white bars) or LNA21-transfected (grey bars) cells. R.f.u.: relative fluorescence units. (D) Time-course analysis of the cell cycle in UNT or oligomer-transfected PCa cells. Data represent mean values ± SD of at least three independent experiments.

Figure 3

Analysis of miR-21 knockdown on the chemosensitivity profiles of PCa cells. (A) Growth inhibition curves of untreated (black triangle), LNAScr (black circle)- or LNA21 (white circle)-transfected PCa cells exposed to increasing concentrations of cisplatin or taxol. Data are reported as percentage of growing cells compared to untreated controls and represent mean values ± SD of at least three independent experiments. (B) Quantification of cells with an apoptotic nuclear morphology by propidium iodide staining of PCa cells transfected with LNAScr (white bars) or LNA21 (grey bars) and exposed to IC₅₀ of cisplatin or taxol. Data are reported as the average (± SD) of the percentage of apoptotic cells in the overall cell population.

Figure 4

Analysis of miR-21 knockdown on the radiosensitivity profiles of PCa cells. (A) Clonogenic survival curves of untreated (black triangle), LNAScr (black circle)- or LNA21 (white circle)-transfected cells calculated on day 12 after exposure to increasing doses (2-8 Gy) of γ- radiation. Data shown on the plots represent the inter-experiment averages (± SD) calculated from at least three intra-experiment averages. (B) Representative immunofluorescence analysis of γ- H2AX induction in LNAScr- or LNA21-treated DU145 cells exposed to γ- radiation (4 Gy). Nuclei were counterstained with 4',6-diamidino-2-phenylindole. Scale bar: 10 μm. Magnification: × 40. NR, no radiation. (C) Quantification of γ- H2AX foci in DU145 (top panel) and PC-3 cells (bottom panel). Data are reported as percentage of γ- H2AX-positive cells in the overall cell population (mean values ± SD).

Figure 5

miR-21 expression in carcinomas and normal prostate tissues. (A) Quantification of miR-21 expression levels (top panel) in carcinomas and matched normal tissues obtained from 36 patients subjected to radical prostatectomy. Data are reported as RQ of miR-21 expression with respect to an internal calibrator (RWPE-1). Analysis of miR-21 expression (bottom panel) reported as Log₁₀ of the ratio tumor/matched normal tissue for each patient (RQ_tumor/RQ_normal). (B) miR-21 expression levels (average RQ ± SD) as a function of nodal status, extraprostatic extension (EPE) of the disease and Gleason score. (C) Representative RT-PCR and western immunoblotting showing the expression of miR-21 validated target genes in untreated and LNAScr- or LNA21-treated DU145 cells, at days 2 and 3 after transfection. (D) Scatterplot showing the lack of a correlation between RQ expression values of miR-21 and PTEN mRNA in clinical specimens.