PCA3 noncoding RNA is involved in the control of prostate-cancer cell survival and modulates androgen receptor signaling

Abstract

Background: PCA3 is a non-coding RNA (ncRNA) that is highly expressed in prostate cancer (PCa) cells, but its functional role is unknown. To investigate its putative function in PCa biology, we used gene expression knockdown by small interference RNA, and also analyzed its involvement in androgen receptor (AR) signaling.

Methods: LNCaP and PC3 cells were used as in vitro models for these functional assays, and three different siRNA sequences were specifically designed to target PCA3 exon 4. Transfected cells were analyzed by real-time qRT-PCR and cell growth, viability, and apoptosis assays. Associations between PCA3 and the androgen-receptor (AR) signaling pathway were investigated by treating LNCaP cells with 100 nM dihydrotestosterone (DHT) and with its antagonist (flutamide), and analyzing the expression of some AR-modulated genes (TMPRSS2, NDRG1, GREB1, PSA, AR, FGF8, CdK1, CdK2 and PMEPA1). PCA3 expression levels were investigated in different cell compartments by using differential centrifugation and qRT-PCR.

Results: LNCaP siPCA3-transfected cells significantly inhibited cell growth and viability, and increased the proportion of cells in the sub G0/G1 phase of the cell cycle and the percentage of pyknotic nuclei, compared to those transfected with scramble siRNA (siSCr)-transfected cells. DHT-treated LNCaP cells induced a significant upregulation of PCA3 expression, which was reversed by flutamide. In siPCA3/LNCaP-transfected cells, the expression of AR target genes was downregulated compared to siSCr-transfected cells. The siPCA3 transfection also counteracted DHT stimulatory effects on the AR signaling cascade, significantly downregulating expression of the AR target gene. Analysis of PCA3 expression in different cell compartments provided evidence that the main functional roles of PCA3 occur in the nuclei and microsomal cell fractions.

Conclusions: Our findings suggest that the ncRNA PCA3 is involved in the control of PCa cell survival, in part through modulating AR signaling, which may raise new possibilities of using PCA3 knockdown as an additional therapeutic strategy for PCa control.

Figure 1

Analysis of gene expression of PCA3 transcript in different cell lines and its targeted knockdown by siPCA3 in PCa cells. (A) RNA expression of PCA3 was quantified by qRT-PCR in different prostate (LNCaP, PrEC, RWPE-1, DU145, and PC3) and non-prostate cell lines (NIH3T3 and HeLa). PCA3 expression was determined using the oligonucleotide primers described in the Methods section. PCA3 relative expression levels were determined in each cell line and compared to PCA3 expression in the DU145 cell line, used as a reference in this assay. (B) PCA3 expression was evaluated in LNCaP cells after knockdown using three different siPCA3 RNA sequences, termed siPCA3/1, siPCA3/2, and siPCA3/3. PCA3 expression was evaluated at 36 h post-transfection, and its relative expression level was determined compared to LNCaP cells transfected with the siSCr sequence, used as a negative control in this assay. (C) Following transfection of LNCaP cells with siPCA3/2, transcript levels were evaluated by qRT-PCR assays at the indicated time points. PCA3 relative expression is shown compared to siScr/LNCaP transfected cells. (D) PC3 cells were also transfected with siPCA3/2, and PCA3 expression was evaluated by qRT-PCR after 36 h post-transfection. 18S RNA was used as a constitutive gene. Data are shown as mean ± SD. All experiments were biological replicates, repeated a minimum of three times. **, p < 0.0017 and *** p < 0.0008.

Figure 2

PCA3 knockdown inhibits LNCaP cell growth and survival. (A) LNCaP cells were transfected with 60 nM of siPCA3 and non-targeting siRNA (siScr), and then followed for cell proliferation for 96 h. (B) NIH3T3 and (C) HeLa cells were transfected with 60 nM of siPCA3 or non-targeting siRNA (siScr) and then followed for cell growth for 48 h. Cell growth was evaluated using the crystal-violet assay described in the Methods section. Data represent the mean ± SD of three independent experiments (* p < 0.05). (D) The effect of PCA3 knockdown on LNCaP cell viability was measured using Trypan Blue exclusion analysis. Data are shown as the mean ± SD of three experiments.

Figure 3

PCA3 knockdown induces cell cycle arrest and apoptosis in LNCaP cells. (A) Representative histograms of LNCaP cells 36 h after transfection with siPCA3 or siScr (left panels). Flow cytometry was used to quantify the percentage of cells undergoing apoptosis (cells in sub G0-G1), which are shown in the bar graph on the right panel. The data represent the means ± S.D. from three independent experiments (right panel). The asterisks indicate significant differences between siPCA3 and siScr control-treated groups (*** p < 0.001). (B) Nuclear morphological changes characteristic of apoptotic cells in LNCaP cell cultures transfected with siPCA3 or siScr (left panels) were analyzed. A representative experiment is shown. Following fixation and staining with DAPI, LNCaP cells transfected with siPCA3 or siScr were examined and photographed using a fluorescence microscope as described in the Methods section. The number of pyknotic nuclei in each experimental sample was counted, and is represented on the bar graph in the right panel as the percentage of cells with pyknotic nuclei in relation to the total number of cells counted. (C) Nuclear morphological changes characteristic of apoptotic cells in PC3 cell cultures transfected with siPCA3 or siScr (left panels) were analyzed, and the results represented as described for LNCaP cells in (B). The data represent the means ± S.D. from three independent experiments (right panel).

Figure 4

PCA3 expression is upregulated by androgen and modulates the transcription of androgen-regulated genes. (A) PCA3 expression was evaluated in LNCaP cells by qRT-PCR after treatment with 100 nM of DHT or 100 nM DHT plus 100 nM flutamide during a 48-h time course, as described in the Methods section. PCA3 relative expression was determined compared to LNCaP cells treated with ethanol, which was the control vehicle. Error bars +/- SD. (B) Relative RNA quantification of PCA3, AR and androgen-regulated genes (TMPRSS2, NDRG1, GREB1, PSA, AR, FGF8, CdK1, CdK2, and PMEPA1) in LNCaP cells treated with 100 nM of dihydrotestosterone (DHT) for 36 h, compared to cells treated with the control vehicle (ethanol), as described in the Methods section. Bar graphs show the average transcript levels of each gene tested, by qRT–PCR analysis of three independent RNA samples prepared following the treatment of LNCaP cells with DHT or ethanol only. Error bars +/- SD. (C) Relative RNA levels of PCA3, AR, and androgen-regulated genes 36 h after LNCaP cells were transfected with siPCA3, compared to LNCaP/siSCr transfected cells. Error bars +/- SD. (D) Relative RNA levels of PCA3, AR, and androgen-regulated genes 36 h after LNCaP cells were transfected with siPCA3 simultaneously with treatment with 100 nM DHT, compared to LNCaP cells transfected with siScr simultaneously with treatment with 100 nM DHT, as described in the Methods section. 18S RNA was used as a constitutive gene in all these assays. Data are represented as mean ± SD. All experiments were biological replicates repeated a minimum of three times. *, p < 0.01, in comparison to scrambled-siRNA (siScr) treated cells.

Figure 5

Downregulation of PCA3 expression does not significantly reduce Akt and Erk1/2 phosphorylation in LNCaP cells. The Akt and Erk1/2 phosphorylation profile before and after transfection of LNCaP cells with either siPCA3 or siScr was investigated by immunoblot analysis using 50 μg of total protein extracts. Akt and Erk1/2 non-phosphorylated proteins were used as controls for normalization of protein loading.

Figure 6

PCA3 is mostly expressed in LNCaP nucleus and microsomal cell fractions. (A) Total RNA samples obtained from different LNCaP cell compartments. Fraction P1 contained mainly nuclei and some vesicles. Fraction P2 contained nuclei, mitochondria, dense bodies, and vesicles. Fraction P3 contained mainly mitochondria, with a few vesicles and dense bodies. Finally, fraction P4 contained free vesicles and microsomes. Bar graphs represent PCA3 ncRNA relative expression levels in each subcellular compartment, compared to total RNA from LNCAP cell whole extracts, containing a mixture of RNA samples from all cell compartments. 18S rRNA was used as a constitutive gene. (B) Analysis of PCA3 expression by RT-PCR in total RNA samples from primary prostate stromal cell cultures. M: molecular weight marker, 100 bp; Lane 1: Total RNA samples from a single PCa tumor expressing PCA3; Lanes 2 – 4: Three different primary prostate stromal-cell cultures.