miR-17-5p targets the p300/CBP-associated factor and modulates androgen receptor transcriptional activity in cultured prostate cancer cells

Abstract

Background: Androgen receptor (AR) signalling is critical to the initiation and progression of prostate cancer (PCa). Transcriptional activity of AR involves chromatin recruitment of co-activators, including the p300/CBP-associated factor (PCAF). Distinct miRNA expression profiles have been identified in PCa cells during the development and progression of the disease. Whether miRNAs regulate PCAF expression in PCa cells to regulate AR transcriptional activity is still unclear.

Methods: Expression of PCAF was investigated in several PCa cell lines by qRT-PCR, Western blot, and immunocytochemistry. The effects of PCAF expression on AR-regulated transcriptional activity and cell growth in PCa cells were determined by chromatin immunoprecipitation, reporter gene construct analysis, and MTS assay. Targeting of PCAF by miR-17-5p was evaluated using the luciferase reporter assay.

Results: PCAF was upregulated in several PCa cell lines. Upregulation of PCAF promoted AR transcriptional activation and cell growth in cultured PCa cells. Expression of PCAF in PCa cells was associated with the downregulation of miR-17-5p. Targeting of the 3'-untranslated region of PCAF mRNA by miR-17-5p caused translational suppression and RNA degradation, and, consequently, modulation of AR transcriptional activity in PCa cells.

Conclusions: PCAF is upregulated in cultured PCa cells, and upregulation of PCAF is associated with the downregulation of miR-17-5p. Targeting of PCAF by miR-17-5p modulates AR transcriptional activity and cell growth in cultured PCa cells.

Figure 1

Expression of PCAF in cultured cell lines. A and B, expression of PCAF as assessed by Western blot and qRT-PCR, respectively. Representative Western blots are shown in A; β-actin was also blotted to ensure equal loading. The amount of PCAF mRNA is shown in B. Data in B are averages of three independent experiments. *, p < 0.05 compared to RWPE1 and PrEC cells. C and D, expression of PCAF protein in RWPE1 and LNCaP cells as assessed by immunofluorescent staining. PCAF was stained in green using a specific anti-PCAF antibody (in D). Cell nuclei were stained in blue with DAPI and overlaid images were also shown. The non-specific IgG was used as the control (as shown in C). Bar = 10 μm.

Figure 2

PCAF is a co-activator to AR and promotes DHT-induced AR transcriptional activity and cell growth. A, knockdown of PCAF by siRNA and forced expression of PCAF through transfection of the pCX-PCAF in LNCaP cells, as confirmed by Western blot and PCR analysis. B, functional manipulation of PCAF on DHT-induced transcription of PSA in LNCaP cells. Cells were exposed to DHT (10 nM) for up to 48 h, followed by qRT-PCR. DHT was also used to stimulate LNCaP cells that were pre-treated with siRNA to PCAF or transfected with the pCX-PCAF for 24 h. C and D, functional manipulation of PCAF on DHT-induced PSA-6 kb luciferase activity in LNCaP cells. Cells were transfected with the PSA-6 kb luciferase reporter plasmid with PCAF siRNA or pCX-PCAF for 24 h, then exposed to DHT (10 nM) for 24 h. Luciferase activity was measured and presented as the ratio to β-gal. E and F, functional manipulation of PCAF on DHT-stimulated LNCaP cell growth. Cells were transfected with pCX-PCAF (E) or treated with PCAF siRNA (F) for 24 h and then exposed to DHT (10 nM) for 72 h, followed by MTS assay. G and H, ChIP analysis of DHT-induced promoter recruitment AR and PCAF to the ARE region of the PSA gene in LNCaP cells. Cells were exposed to DHT (10 nM) for 5 h and immunoprecipitated with antibodies to AR or PCAF, or the IgG control. Specific PCR primers covering the ARE-I region of the PSA promoter were used for the PCR analysis and data were presented as ratio to the input. Data in A to F are averages of three independent experiments. *, p < 0.05 compared to non-DHT treated cells (in B, C, D, E, F and H) or siRNA control (in A). ^#, p < 0.05 compared to cells treated with DHT only. Luc = luciferase activity.

Figure 3

miR-17-5p targets PCAF 3’UTR, resulting in translational suppression and RNA degradation. A, the schematic of human PCAF mRNA showed a potential binding site in its 3’UTR for miR-17-5p. The complementary miR-17-5p-binding site in the PCAF 3’UTR was inserted to the downstream of a luciferase reporter on the pMIR-REPORT plasmid. A control plasmid with the mutant 3’UTR sequence was also generated. WT = wild-type. B, binding of miR-17-5p to the potential binding site in the PCAF 3’UTR results in translational suppression, as assessed by luciferase reporter assay. LNCaP and RWPE1 cells were transfected with the plasmids and treated with the anti-miR or precursor to miR-17-5p, or non-specific oligo control, for 24 h, followed by luciferase analysis. Mut = mutant; *, p < 0.05 compared to the controls; #, p < 0.05 compared to PCAF 3’UTR transfected alone. C, expression of miR-17-5p in cells, as assessed by qRT-CR. Mature miR-17-5p level was obtained by normalizing to the endogenous reference RNU6B. D and E, miR-17-5p precursor decreases PCAF expression in LNCaP cells. Cells were treated with various doses of miR-17-5p precursor or nonspecific precursor control, followed by Western blot for PCAF protein (after incubation for 48 h) or PCR for PCAF mRNA (after incubation for 24 h). F and G, anti-miR-17-5p increases PCAF expression in RWPE1 and LNCaP cells. Cells were treated with various doses of anti-miR-17-5p or non-specific anti-miR control followed by Western blot for PCAF protein (48 h) or PCR for PCAF mRNA (24 h). Data in B, C, E, and G are averages of three independent experiments. *, p < 0.05 compared to RWPE1 and PrEC cells (in C) or the controls in E and G.

Figure 4

Effects of functional manipulation of miR-17-5p on DHT-induced AR transcriptional activity and cell growth.A and B, impact of functional manipulation of miR-17-5p on DHT-induced transcription of the PSA gene. LNCaP cells were treated with anti-miR-17-5p or miR-17-5p precursor for 48 h and then exposed to DHT (10 nM) for an additional 24 h, followed by qRT-PCR analysis. C - F, impact of functional manipulation of miR-17-5p on DHT-induced PSA-6 kb luciferase activity in LNCaP cells (C and D) and C4-2B cells (E and F). Cells were transfected with the PSA-6 kb luciferase reporter plasmid and simultaneously treated with anti-miR-17-5p or miR-17-5p precursor for 48 h, then exposed to DHT (10 nM) for an additional 24 h. Luciferase activity was measured and presented as the ratio to β-gal. G, inhibition of miR-17-5p precursor on DHT-stimulated LNCaP cell growth. Cells were treated with miR-17-5p precursor for 48 h then exposed to DHT (10 nM) for 72 h, followed by MTS assay. Data are averages of three independent experiments. *, p < 0.05 compared to non-DHT-treated cells (in A -G); ^#, p < 0.05 compared to cells treated with DHT only. Luc = luciferase activity.