Rho GTPase activating protein 21-mediated regulation of prostate cancer associated 3 gene in prostate cancer cell

Abstract

The overexpression of the prostate cancer antigen 3 (PCA3) gene is well-defined as a marker for prostate cancer (PCa) diagnosis. Although widely used in clinical research, PCA3 molecular mechanisms remain unknown. Herein we used phage display technology to identify putative molecules that bind to the promoter region of PCA3 gene and regulate its expression. The most frequent peptide PCA3p1 (80%) was similar to the Rho GTPase activating protein 21 (ARHGAP21) and its binding affinity was confirmed using Phage Bead ELISA. We showed that ARHGAP21 silencing in LNCaP prostate cancer cells decreased PCA3 and androgen receptor (AR) transcriptional levels and increased prune homolog 2 (PRUNE2) coding gene expression, indicating effective involvement of ARHGAP21 in androgen-dependent tumor pathway. Chromatin immunoprecipitation assay confirmed the interaction between PCA3 promoter region and ARHGAP21. This is the first study that described the role of ARHGAP21 in regulating the PCA3 gene under the androgenic pathway, standing out as a new mechanism of gene regulatory control during prostatic oncogenesis.

Figure 1. Prostate cancer associated 3 (PCA3) amplification in peripheral blood samples. A, Representation of qualitative RT-Nested PCR of PCA3 gene. B, Clinicopathological data of selected patients (n=26 patients); 24 PCa (1-17, 10-21, and 23-26) and 2 BPH (18 and 22). C, Amplification of PCA3 promoter segment with the biotinylated sense primer. M: 100-pb ladder; NC: negative control. PCa: prostate cancer; BPH: benign prostatic hyperplasia; TNM: tumor, node, metastasis; PSA: prostate-specific antigen.

Figure 2. Sequence and alignment of PCA3p1 and PCA3p2. ARHGAP21: Rho GTPase activating protein 21; CYP3A4: Cytochrome P450 3A4.

Figure 3. Basal transcriptional levels of prostate cancer associated 3 (PCA3), Rho GTPase activating protein 21 (ARHGAP21), prune homolog 2 (PRUNE2), and androgen receptor (AR) in RWPE-1, LNCaP, and PC-3 cells. PCA3 transcripts were upregulated in LNCaP cells, the best model to study this lncRNA pathway. *P<0.05, **P<0.01 (ANOVA).

Figure 4. Rho GTPase activating protein 21 (ARHGAP21) binds to PCA3 promoter region (PCA3prom) and regulates its transcripts. A, Chromatin immunoprecipitation (Chip) and iRNA assays were performed to confirm the regulation of prostate cancer associated 3 (PCA3) mediated by endogenous ARHGAP21. Chip was carried out with anti-ARHGAP21 (ARHGAP21) or anti-EGFR antibody (EGFR) followed by PCA3 promoter and nitric oxide synthase 3 (NOS3) amplification. DNA extracted from LNCaP cells (LNCaP) and LNCaP cell lysate (input) were used as controls. Chip assay was also performed in the absence of cellular material (Blank). B, RT-qPCR quantification of LNCaP cells silenced for ARHGAP21 (esiARHGAP21) compared to cells subjected to silencing of the irrelevant gene, GFP (esiGFP). C, Western blotting analysis of ARHGAP21 (250 kDa) expression in LNCaP cells. The image shows the ARHGAP21 silencing (esiARHGAP21) compared to the untreated cells (Crtl) and the cells subjected to silencing of the irrelevant gene, GFP (esiGFP). Lamin B was used as a loading control of protein extracts. D, Quantification of western blotting image by ImageJ software (USA). Three independent stainings were performed. E, Transcriptional levels of PCA3 in the LNCaP cells silenced for ARHGAP21 (esiARHGAP21) compared to the cells subjected to silencing of the irrelevant gene, GFP (esiGFP). F, mRNA relative expression levels of PRUNE2 and (G) AR were recorded in LNCaP-ARHGAP21 silenced cells. bp: base pairs; kDa: kilodaltons. Data are reported as means±SD. *P<0.05, ***P<0.001, ****P<0.0001 (Student's t-test and ANOVA).

Figure 5. Phage binding analysis. A, Bead-ELISA performed with a biotinylated PCA3 promoter amplicon and streptavidin-conjugated magnetic beads. The assay was carried out with PCA3p1 phage (PCA3p1) and wild-type phage that do not display any peptide (wild-type). Wells without phage particles (blank) were included as control. qPCR assays were also performed after the treatment of prostate cells with PCA3p1. B, Transcriptional levels of PCA3 (prostate cancer associated 3), (C) ARHGAP21 (Rho GTPase activating protein 21), (D) AR (androgen receptor), and (E) PRUNE2 (prune homolog 2) genes quantified upon cell stimuli with 10⁷ phage particles of PCA3p1 for 48 h. Data are reported as means±SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA).

Figure 6. In silico predictions of the interaction between the ARHGAP21 protein and the peptide PCA3p1 to the promoter region of the PCA3 gene. A, Overview of the peptide PCA3p1 (HGPNIDHLATLH) (cyan), expressed on the surface of the M13 phage and the PCA3prom (green). B, Zoom of the binding interaction between PCA3p1 and PCA3 promoter region. C, Docking overview between the ARHGAP21 (wheat) and PCA3prom. D, Identification of ARHGAP21 binding residues (wheat) and interaction sites onto PCA3prom. E, Structural alignment between PCA3p1 and ARHGAP21. F, Enlarged image of interaction sites between the PCA3p1 peptide and ARHGAP21 with DNA and homology between the binding sites. Polar contacts are shown as dashed yellow lines, and the interaction residues are marked in orange.