miR-331-3p regulates ERBB-2 expression and androgen receptor signaling in prostate cancer

Abstract

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and are aberrantly expressed in human cancer. The ERBB-2 tyrosine kinase receptor is frequently overexpressed in prostate cancer and is associated with disease progression and poor survival. We have identified two specific miR-331-3p target sites within the ERBB-2 mRNA 3'-untranslated region and show that miR-331-3p expression is decreased in prostate cancer tissue relative to normal adjacent prostate tissue. Transfection of multiple prostate cancer cell lines with miR-331-3p reduced ERBB-2 mRNA and protein expression and blocked downstream phosphatidylinositol 3-kinase/AKT signaling. Furthermore, miR-331-3p transfection blocked the androgen receptor signaling pathway in prostate cancer cells, reducing activity of an androgen-stimulated prostate-specific antigen promoter and blocking prostate-specific antigen expression. Our findings provide insight into the regulation of ERBB-2 expression in cancer and suggest that miR-331-3p has the capacity to regulate signaling pathways critical to the development and progression of prostate cancer cells.

Figures

FIGURE 1.

Identification of two specific miR-331-3p target sites within the ERBB-2 mRNA 3′-UTR. A, schematic representation of the ERBB-2 mRNA with two 3′-UTR miR-331-3p binding sites (A and B) predicted by TargetScan. The miR-331-3p seed sequence is underlined. B, sequence alignment of the predicted ERBB-2 3′-UTR miR-331-3p target sites showing conservation between human, mouse, rat, and dog. The miR-331-3p seed sequence (CCAGGGG) is shown in bold and underlined, and conserved nucleotides are shaded. Stars indicate nucleotides conserved across all four species.

FIGURE 2.

miR-331-3p regulates ERBB-2 expression in prostate cancer cell lines. A, immunoblotting detection of ERBB-2 and β-actin expression using protein extracts harvested from LNCaP, 22RV1, and DU145 cells 3 days after transfection with miR-331-3p or the miR-NC precursor. B, qRT-PCR analysis of ERBB-2 mRNA expression in LNCaP, 22RV1, and DU145 cells 24 h after transfection with miR-331-3p or miR-NC. ERBB-2 RNA expression was normalized to GAPDH RNA expression, and is shown as a ratio of miR-331-3p-transfected cells to miR-NC-transfected cells using the 2−ΔΔCT method and GenEx statistical software. Data are representative of three independent experiments. Asterisk indicates a significant difference from miR-NC-transfected control cells (p < 0.03). Error bars represent confidence intervals (CI = 0.95).

FIGURE 3.

miR-331-3p expression is reduced in prostate tumor relative to normal adjacent tissue and is inversely correlated with ERBB-2 mRNA expression. A, qRT-PCR analysis of the pri-miR-331-3p expression in normal adjacent prostate tissue (NAT) RNA versus prostate tumor (T) RNA. Total RNA was reverse transcribed and miR-331-3p expression determined by qRT-PCR. Data were normalized to GAPDH expression and relative tumor miR-331-3p expression was calculated. Asterisk indicates a significant difference between pri-miR-331-3p expression in NAT versus tumor (p < 0.0001). B, qRT-PCR analysis for mature miR-331-3p in NAT versus tumor. Total RNA was reverse transcribed and miR-331-3p expression determined by the TaqMan miRNA qRT-PCR assay. Data were normalized to U44 and U6 small nuclear RNA expression and relative miR-331-3p expression was calculated. Asterisk indicates a significant difference between mature miR-331-3p expression in tumor versus NAT (p < 0.00001). C, qRT-PCR analysis of ERBB-2 mRNA expression in NAT versus tumor RNA. Total RNA was reverse transcribed and ERBB-2 and GAPDH expression determined by qRT-PCR. Data were normalized to GAPDH RNA expression and tumor ERBB-2 was expressed relative to NAT ERBB-2. Asterisk indicates a significant difference between ERBB-2 expression in tumor versus NAT (p < 0.0001). Error bars are as described in the legend to Fig. 2.

FIGURE 4.

The 3′-UTR of ERBB-2 mRNA is a direct target of miR-331-3p via two miR-331-3p target sites. A, schematic representation of firefly luciferase reporter constructs for full-length, wild type ERBB-2 3′-UTR and perfect miR-331-3p target. 22RV1 cells were co-transfected with pmiR-REPORT constructs and miR-NC (1 nm), miR-331-3p (1 nm), LNA-NC (10 nm), or LNA-miR-331-3p (10 nm). B, schematic representation of firefly luciferase reporter constructs of wild type and mutant ERBB-2 3′-UTR miR-331-3p-A and -B target sites and perfect -3p target. 22RV1 cells were co-transfected with pmiR-REPORT and CMV-Renilla constructs, and miR-NC or miR-331-3p (1 nm), and assayed for firefly and Renilla luciferase activities after 24 h. Relative luciferase expression values are expressed as a ratio of miR-NC to LNA. Asterisk indicates significant difference between miR-NC transfected control cells (p < 0.05). All data are representative of at least three independent experiments. Error bars are as described in the legend to Fig. 2.

FIGURE 5.

miR-331-3p decreases ERBB-2 protein expression and signaling, and blocks PSA expression and promoter activity in LNCaP cells. A, LNCaP cells were transfected with miR-NC or miR-331-3p (30 nm) for 48 h and serum starved for 24 h thereafter, followed by stimulation ± heregulin (HRG; 50 ng/ml) for 20 min. Cell lysates were analyzed for total ERBB-2, phospho-ERBB-2, AR, total AKT, and phospho-AKT expression by immunoblotting. B, LNCaP cells were transfected with miR-331-3p for 48 h and treated ± DHT (10 nm) and ± bicalutamide (10 μm). Total PSA expression was determined by immunoblotting. C, LNCaP cells were co-transfected with a PSA-luciferase vector (PSA-LUC) and thymidine kinase-Renilla vector and with miR-NC or miR-331-3p (1 nm). Relative luciferase expression (firefly normalized to Renilla) values are expressed as a ratio of miR-NC-transfected cells (±S.D.). Asterisk indicates significant difference between miR-NC transfected control cells (p < 0.05). Error bars represent confidence intervals (CI = 0.95).

FIGURE 6.

miR-331-3p blocks AR signaling via inhibition of ERBB-2 expression and AKT activity in prostate cancer cells. AR antagonists such as bicalutamide bind to the AR and prevent its activation and expression of AR target genes, such as PSA. Nevertheless, AR signaling may persist in prostate cancer cells despite AR blockade, in part via increased expression of the ERBB-2 receptor tyrosine kinase and subsequent activation of the PI3K/AKT pathway, which causes AR phosphorylation and promotes expression of AR target genes. miR-331-3p directly targets the ERBB-2 mRNA 3′-UTR to regulate ERBB-2 protein expression, thereby reducing PI3K/AKT signaling and AR signaling. The combination of an AR antagonist (bicalutamide) and miR-331-3p effectively blocks AR signaling (PSA expression and PSA promoter activity) in LNCaP prostate cancer cells. (+) indicates activation step of pathway and (−) indicates inhibition of pathway component.