Upregulation of miR-3195, miR-3687 and miR-4417 is associated with castration-resistant prostate cancer

Abstract

Purpose: Prostate cancer (PCa) is a leading cause of cancer-related death. Upon androgen-deprivation therapy, the disease may progress further to castration-resistant PCa (CRPC) with a poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs, which play crucial roles in gene regulation. The aim of our study is to find CRPC-associated miRNAs and to evaluate their functional role.

Methods: In this study, 23 benign prostatic hyperplasia (BPH), 76 primary PCa, and 35 CRPC specimens were included. Total RNA extracted from tissue sections was used for miRNA profiling on the Affymetrix GSC 3000 platform. Subsequently, stem-loop RT-qPCR analysis was performed to validate the expression levels of selected miRNAs. PCa cell lines were transfected with miRNA mimics or inhibitors to evaluate the effects on cell proliferation, cell migration and cell invasion.

Results: In our profiling study, several miRNAs were found to be deregulated in CRPC compared to primary PCa tissue, of which miR-205 (- 4.5-fold; p = 0.0009), miR-92b (- 3.1 fold; p < 0.0001) were downregulated and miR-3195 (5.6-fold; p < 0.0001), miR-3687 (8.7-fold; p = 0.0006) and miR-4417 (5.0-fold; p = 0.0005) were most upregulated. While KLK3, miR-21 and miR-141 expression levels in androgen-treated VCaP and LNCaP cells were increased, the expression levels of miR-3687 and miR-4417 were reduced. None of the miRNAs were androgen-regulated in the AR-negative PC3 cell line. Overexpression of miR-3687 reduced cell migration and cell invasion, whilst miR-3195 enhanced cell migration.

Conclusion: We have identified several novel deregulated miRNAs in CRPC tissue, including two microRNAs that are potentially involved in tumor invasion. Our data support the hypothesized involvement of miRNAs in PCa tumorigenesis and progression to CRPC. The applicability of these miRNAs as novel biomarkers for CRPC remains to be further investigated.

Keywords: Androgens; Castration-resistance; MicroRNA; Prostate cancer.

Fig. 1

Expression of miR-205, miR-3195, miR-3687, and miR-4417 in human prostate tissue and their association with CRPC. MiRNA expression analysis was performed by stem-loop RT-qPCR analysis. The results were normalized to the weighted average of miR-26a, miR-107 and RNU6 levels. The median expression and the standard deviation are shown; outliers are indicated by open circles and asterisks. MiR-205 a is significantly downregulated, whilst miR-3195 b, miR-3687 c and miR-4417 d are significantly upregulated in CRPC tissue compared to primary PCa tissue. BPH, benign prostate hyperplasia; LG, low grade prostate cancer; HG, high grade prostate cancer; CRPC, castration-resistant prostate cancer. e, f Receiver operating characteristic curve (ROC) analysis was performed using miRNA expression data from the validation set (n = 83), comparing the ability of miR-3195, miR-3687, miR-4417 alone and combinations of miR-3195 and miR-3687, and of miR-205, miR-3195 and miR-4417 to identify men with CRPC. Areas under the curve (AUC) are shown (*p < 0.01)

Fig. 2

Androgen responsiveness of CRPC-associated miRNAs. Analysis of KLK3 a, miR-21 b, miR-141 c, miR-3195 d, miR-3687 e and miR-4417 f expression in VCaP cells. VCaP cells were cultured for 3 days in androgen-free (CSS) medium, and then incubated in the presence of 0.1, 1 or 10 nM of synthetic androgen R1881. Total RNA was extracted 24 and 48 h after androgen stimulation. KLK3 and miRNA levels were determined by reverse transcriptase real-time PCR analysis. HPRT1 and RNU6 levels were used for normalization purposes. All experiments were repeated at least three times; p-values were calculated with the unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 3

Effect of CRPC-associated miRNAs on cell survival. PC3 a–c, f and VCaP d–e cells were transfected with miRNA mimics (10–50 nM) or with anti-miR miRNA inhibitors (50 nM). Total RNA was extracted and the relative expression of miR-3687 was determined by stem-loop RT-qPCR analysis, using RNU6 for normalization a, d. Cell viability of miRNA mimic or anti-miR transfected cells was determined at different time points post-transfection b, c, e. Transfected cells were seeded 2 days post-transfection in 96-well plates. Cell viability was determined from day 1 until day 5 (PC3) or day 11 (VCaP), using CellTiter-Glo Assays. Apoptosis of miRNA mimic and anti-miR transfected PC3 cells was determined 2 days post transfection, using a Caspase-3/7 Assay f. NT non-transfected cells. Error bars indicate the standard deviation of at least 2 independent experiments

Fig. 4

Effect of CRPC miRNAs on cell migration and cell invasion. Cell migration a was assessed by wound-healing “scratch” assays. Error bars indicate the standard deviation of at least four independent experiments performed in triplicate. Cell invasion b was determined using Matrigel-coated 8-µm cell invasion chambers. PC3 cells were transfected with miRNA mimics (10–50 nM) or with anti-miR miRNA inhibitors. Inhibition of cell invasion by a specific miRNA was calculated by dividing the percentage of invaded cells versus that of NC2 transfected cells. NT non-transfected cells. Error bars indicate the standard deviation of two independent experiments performed in duplicate; p values were calculated with the unpaired t-test (*p < 0.05; ***p < 0.001)