MiR-193b promoter methylation accurately detects prostate cancer in urine sediments and miR-34b/c or miR-129-2 promoter methylation define subsets of clinically aggressive tumors

Background: Contemporary challenges of prostate cancer (PCa) include overdiagnosis and overtreatment, entailing the need for novel clinical tools to improve risk stratification and therapy selection. PCa diagnosis and prognostication might be perfected using epigenetic biomarkers, among which aberrant DNA methylation of microRNA promoters has not been systematically explored. Herein, we identified aberrantly methylated microRNAs promoters in PCa and assessed its diagnostic and prognostic biomarker potential. Methods: Using HumanMethylation450 BeadChip-based analysis differentially methylated CpGs in microRNA promoters were identified. Promoter methylation of six microRNAs (miR-34b/c, miR-129-2, miR-152, miR-193b, miR-663a and miR-1258) was analyzed by qMSP in three sets (180 prostatectomies, 95 urine sediments and 74 prostate biopsies). Biomarkers’ diagnostic (validity estimates) and prognostic [disease-free (DFS) and disease-specific survival (DSS)] performance was assessed. Results: Significantly higher promoter methylation levels in PCa were confirmed for six candidate microRNAs. Except for miR-152, all displayed AUC values higher than 0.90, with miR-1258 and miR-193b disclosing the best performance (AUC = 0.99 and AUC = 0.96, respectively). In urine samples, miR-193b showed the best performance (91.6% sensitivity, 95.7% specificity, AUC = 0.96). Moreover, higher miR-129-2 independently predicted for shorter DSS and miR−34b/c methylation levels independently predicted for shorter DFS and DSS. Conclusions: Quantitative miR-193b, miR-129-2 and miR-34b/c promoter methylation might be clinically useful PCa biomarkers for non-invasive detection/diagnosis and prognostication, both in tissue and urine samples.The authors would like to acknowledge funding attributed to this study, namely research grants from Research Center of Portuguese Oncology Institute of Porto (CI-IPOP 4–2012; CI-IPOP 19–2016)) and by Federal funds through Programa Operacional Temático Factores de Competitividade (COMPETE) with co-participation from the European Community Fund (FEDER) and by national funds through Fundação para a Ciência e Tecnología (FCT) under the projects EXPL/BIM-ONC/0556/2012. JRC was supported by a FCT-Fundação para a Ciência e a Tecnologia fellowship (SFRH/BD/71293/2010)

Additional file 2: Figure S1. of Downregulation of miR-130b~301b cluster is mediated by aberrant promoter methylation and impairs cellular senescence in prostate cancer

DNA methylation changes in microRNAs´ promoters in prostate cancer (PCa), determined by Infinium HumanMethylation450 BeadChip in 25 PCa tissues and 5 morphologically normal prostate tissue (MNPT) samples. (A) Schematic representation of the DNA methylation mapping approach used to identify new aberrantly methylated miRNAs. (B) β values from representative microRNAs. (C) Genomic location of the differentially methylated microRNAs. (D) DNA methylation levels determined by pyrosequencing in PCa cell lines, showing that all cell lines tested display aberrant DNA methylation in the promoter of miR-130b~301b cluster. (*p < 0.05, **p < 0.01, ***p < 0.001). Figure S2. Confirmation of miR-130b and miR-301b expression levels by RT-qPCR. (A–C) miR-130b or miR-301b expression levels after transfection with anti-miR-NC, anti-miR-130b and, anti-miR-301b in LNCaP, DU145 and, PC3, respectively. (D) Overexpression of miR-130b and miR-310b in PC3 cells. The analyses were conducted 72 h post-transfections. All data are presented as mean of three independent experiments ± s.d. (*p < 0.05, **p < 0.01, ***p < 0.001). Figure S3. Cross-validation of deregulated genes upon cluster miR-130b-301b manipulation in the TCGA cohort. Boxplot depiction of the cancer versus normal differentially expressed mRNAs among the TCGA prostate RNA-seq cohort. Green and red squares refer to down-regulated and overexpressed genes in PCa versus NAT samples, respectively. Each point represents one RNA-seq tissue sample. Figure S4. RT-qPCR expression changes in multiple genes involved in invasion and epithelial to mesenchymal transition (EMT), suggesting functional specialization among members of miR-130b-301b polycistron. Gene expression patterns by (A) induction of pre-miR-130b or pre-miR-301b or (B) after endogenous levels blocking. The analyses were conducted 72 h post-transfections. All data is presented as mean of three independent experiments ± s.d. (*p < 0.05, **p < 0.01, ***p < 0.001). Figure S5. Morphological alterations in PC3 cells after miR-130b or miR-301b overexpression. Transfection of (A) pre-miR-NC, (B) pre-miR-130b or (C) pre-miR-301b. The restoration of miR-130b or miR-301b expression induced cell polarization and epithelial-like phenotype, suggesting a mesenchymal to epithelial transition. Figure S6. Morphological alterations in PC3 cells upon miR-130b or miR-301b knockdown. (A) PC3 cells transfected with anti-miR-NC, (B) anti-miR-130b or (C) anti-miR-301b. Inhibition of endogenous miR-130b or miR-301b caused PC3 cells to acquire a more pronounced fibroblast-like morphology, compatible with a mesenchymal-type phenotype. Figure S7. LMNB1 3′ UTR putative binding sites for miR-130b and miR-301b. (PDF 2147 kb

RT-PCR analysis.

<p>Uromodulin (distal tubule marker), aquaporin 3 (collecting duct marker), and aminopeptidase A (proximal tubule marker) in primary cultured HPTEC derived from 4 donors and HK-2 cells. RNA isolated from a mixture of renal cells was used as a positive control and water as a negative control. Expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a house-keeping gene.</p

Schematic representation of the isolation procedure for HPTEC and HRTC.

<p>Schematic representation of the isolation procedure for HPTEC and HRTC.</p

Representative morphology of confluent monolayers.

<p>(A) Primary HPTEC, (B) HK-2 cell line, (C) primary clear cell RCC, and (D) primary chromophobe RCC. Magnification: 400x.</p

Fluorescence microscopy images from immunocytochemical staining for cytokeratin.

<p>Hoechst 33258, anti-cytokeratin antibody staining, and merged images with double staining of primary cultured HPTEC, clear cell and chromophobe RCC cells. HK-2 and A-498 cells were used as control of human normal and tumor epithelial kidney cells, respectively. Magnification: 200x.</p

Donors characteristics and respective isolates parameters.

<p>Donors characteristics and respective isolates parameters.</p

Representative histological images of renal cell carcinoma.

<p>(A) Clear cell renal cell carcinoma, and (B) chromophobe renal cell carcinoma.</p

RPS19, RPS21, and RPS24 protein expression using immunofluorescence.

<p>Representative images of the co-expression of RPS19 (Cy3- Red), RPS21 (FITC- Green), and RPS24 (Cy5- Blue) which was analysed in both malignant (D, E, F) and non-malignant (A, B, C) prostate tissue. Whole tissue cores (A and D) were imaged on an Olympus IX81 confocal system at low magnification. Intensity of fluorescent signals were optimised at the beginning of each study to prevent oversaturation for each fluorophore. Quantification of co-localization was carried out on images gathered from a randomly selected area of the tissue core using a 40x objective zoom (E is malignant and B is non-malignant) on an Olympus IX81 confocal system. The image was further magnified using a 6x digital zoom. Each image was then deconvolved (C and F) and the GIC was calculated using Huygens software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186047#sec003" target="_blank">Materials and methods</a>). Scale bar = 10 μm.</p

Box plots of co-localization of RPS proteins.

<p>Global intersection coefficient (GIC) was calculated using deconvolved, high magnification, fluorescent images (represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186047#pone.0186047.g005" target="_blank">Fig 5</a>) using Huygens software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186047#sec003" target="_blank">Materials and methods</a>). Box plots of GIC were created for comparison between malignant (red bordered bars) and non-malignant (green bordered bars) co-localization for RPS21/RPS19 (green/red bars), RPS21/RPS24 (green/blue bars), and RPS19/RPS24 (red/blue bars). Significance of the difference in GIC values for malignant vs non-malignant prostate tissue was calculated using the Mann Whitney U test (* = <i>p</i><0.01).</p