Role of a novel race-related tumor suppressor microRNA located in frequently deleted chromosomal locus 8p21 in prostate cancer progression

Abstract

The prostate cancer (PCa) genome is characterized by deletions of chromosome 8p21-22 region that increase significantly with tumor grade and are associated with poor prognosis. We proposed and validated a novel, paradigm-shifting hypothesis that this region is associated with a set of microRNA genes-miR-3622, miR-3622b, miR-383-that are lost in PCa and play important mechanistic roles in PCa progression and metastasis. Extending our hypothesis, in this study, we evaluated the role of a microRNA gene located in chromosome 8p-miR-4288-by employing clinical samples and cell lines. Our data suggests that (i) miR-4288 is widely downregulated in primary prostate tumors and cell lines; (ii) miR-4288 expression is lost in metastatic castration-resistant PCa; (ii) miR-4288 downregulation is race-related PCa alteration that is prevalent in Caucasian patients and not in African Americans; (iii) in Caucasians, miR-4288 was found to be associated with increasing tumor grade and high serum prostate-specific antigen, suggesting that miR-4288 downregulation/loss may be associated with tumor progression specifically in Caucasians; (iv) miR-4288 possess significant potential as a molecular biomarker to predict aggressiveness/metastasis; and (v) miR-4288 is anti-proliferative, is anti-invasive and inhibits epithelial-to-mesenchymal transition; and (vi) miR-4288 directly represses expression of metastasis/invasion-associated genes MMP16 and ROCK1. Thus, the present study demonstrates a tumor suppressor role for a novel miRNA located with a frequently lost region in PCa, strengthening our hypothesis that this locus is causally related to PCa disease progression via loss of microRNA genes. Our study suggests that miR-4288 may be a novel biomarker and therapeutic target, particularly in Caucasians.

Figure 1.

MicroRNA-4288 located in frequently deleted chr8p21 region is underexpressed in PCa. (A) Relative miR-4288 expression levels in microdissected prostate tumor tissues (n = 74) and matched adjacent normal tissues as assessed by real-time PCR. Lower panel: Average miR-4288 expression in normal and tumor tissues. (B) Relative miR-4288 expression levels in prostate cell lines as assessed by RT-PCR. Data were normalized to RNU48 control. Data are represented as mean ± SEM (*P < 0.05). (C) ROC curve analysis showing the ability of miR-4288 expression to discriminate between tumor and normal samples.

Figure 2.

miR-4288 downregulation is a race-related PCa alteration associated with increasing tumor grade and high serum PSA in Caucasians. (A) Correlation of miR-4288 expression with clinicopathological characteristics of PCa patients. (B) Kaplan–Meir survival analyses of PCa patients stratified based on miR-4288 expression levels. (C) Relative miR-4288 expression levels in prostate cell lines as assessed by RT-PCR. (D) Correlation of miR-4288 expression with clinicopathological characteristics in Caucasians versus AA PCa patients. P-values are based on chi-square test.

Figure 3.

MicroRNA-4288 is expression is highly downregulated in metastatic CRPC tissues. (A) Relative miR-4288 expression in microdissected metastatic CRPC tumor tissues (n = 33) when compared with normals (n = 19) as assessed by real-time PCR. (B) ROC curve analysis showing the ability of miR-4288 expression to discriminate between metastatic tumor and normal samples.

Figure 4.

miR-4288 overexpression suppresses proliferation of PCa cell lines. Control miRNA (miR-CON)/miR-4288 precursor was transfected in LNCaP/PC3 cell lines at a final concentration of 50nM followed by functional assays (performed 72 h post-transfection). (A) Relative miR-4288 expression after miRNA transfections as assessed by RT-PCR. Data were normalized to RNU48 control. (*P < 0.05). (B) Relative cellular viabilities at the indicated time points after transient transfections as assessed by MTS cellular viability assay. (C) Cell cycle analyses in LNCaP (upper panels)/PC3 cells (lower panels) after miR-CON (left panels) or miR-4288 (right panels) transfections. (D) BrdU cell proliferation assay upon control/miR-4288 expression in LNCaP and PC3 cells. (E) Western blot analyses for indicated proteins after miR-CON/miR-4288 transfection in PC3 cells. GAPDH was used as a loading control.

Figure 5.

miR-4288 expression reduces invasiveness of PCa cell lines and induces mesenchymal to epithelial transition. (A) Trans-well invasion assay in miR-CON/miR-4288-transfected LNCaP and PC3 cells. (B) Morphological alterations upon miR-CON/miR-4288 transfections as assessed by fluorescein isothiocyanate-labeled phalloidin staining in LNCaP and PC3 cells 72 h post-transfection. (C) Relative miR-4288 expression in PC3 cells stably transfected with control miR/miR-4288 overexpression construct as assessed by RT-PCR. Data were normalized to RNU48 control. (D) Upper panel: Real-time PCR analyses of relative transcript levels of CDH1, CDH2 and VIM in PC3 cells stably transfected with miR-CON/miR-4288 overexpression construct. Data were normalized to GAPDH. Lower panel: Immunoblots of endogenous E-cadherin and vimentin in PC3 cells stably transfected with miR-CON/miR-4288.

Figure 6.

miR-4288 directly regulates metastasis-associated genes. (A) Immunoblots for indicated proteins in PC3 and LNCaP cells treated with miR-CON/miR-4288. GAPDH was used as a loading control. (B) Schematic representation of SLUG, ROCK1 and MMP16 3′-UTRs showing putative miR-4288 target sites. (C) Luciferase reporter assays with the indicated wt and mutated 3′-UTR constructs or control luciferase construct co-transfected with miR-CON/miR-4288 in PC3 (left panels) and LNCaP cells (right panels). Firefly luciferase values were normalized to Renilla luciferase activity and plotted as relative luciferase activity (*P < 0.05 when compared with miR-CON).