High-Throughput MicroRNA (miRNAs) Arrays Unravel the Prognostic Role of MiR-211 in Pancreatic Cancer

BACKGROUND: Only a subset of radically resected pancreatic ductal adenocarcinoma (PDAC) patients benefit from chemotherapy, and identification of prognostic factors is warranted. Recently miRNAs emerged as diagnostic biomarkers and innovative therapeutic targets, while high-throughput arrays are opening new opportunities to evaluate whether they can predict clinical outcome. The present study evaluated whether comprehensive miRNA expression profiling correlated with overall survival (OS) in resected PDAC patients. METHODOLOGY/PRINCIPAL FINDINGS: High-resolution miRNA profiles were obtained with the Toray's 3D-Gene™-miRNA-chip, detecting more than 1200 human miRNAs. RNA was successfully isolated from paraffin-embedded primary tumors of 19 out of 26 stage-pT3N1 homogeneously treated patients (adjuvant gemcitabine 1000 mg/m(2)/day, days-1/8/15, every 28 days), carefully selected according to their outcome (OS<12 (N = 13) vs. OS>30 months (N = 6), i.e. short/long-OS). Highly stringent statistics included t-test, distance matrix with Spearman-ranked correlation, and iterative approaches. Unsupervised hierarchical analysis revealed that PDACs clustered according to their short/long-OS classification, while the feature selection algorithm RELIEF identified the top 4 discriminating miRNAs between the two groups. These miRNAs target more than 1500 transcripts, including 169 targeted by two or more. MiR-211 emerged as the best discriminating miRNA, with significantly higher expression in long- vs. short-OS patients. The expression of this miRNA was subsequently assessed by quantitative-PCR in an independent cohort of laser-microdissected PDACs from 60 resected patients treated with the same gemcitabine regimen. Patients with low miR-211 expression according to median value had a significantly shorter median OS (14.8, 95%CI = 13.1-16.5, vs. 25.7 months, 95%CI = 16.2-35.1, log-rank-P = 0.004). Multivariate analysis demonstrated that low miR-211 expression was an independent factor of poor prognosis (hazard ratio 2.3, P = 0.03) after adjusting for all the factors influencing outcome. CONCLUSIONS/SIGNIFICANCE: Through comprehensive microarray analysis and PCR validation we identified miR-211 as a prognostic factor in resected PDAC. These results prompt further prospective studies and research on the biological role of miR-211 in PDAC

Factors associated with OS and DFS in the multivariate analysis.

<p>Abbreviations: df, degrees of freedom; DFS, Disease Free Survival; OS, Overall Survival.</p

Heatmap of the clustering of 170 miRs filtered based on t-test p-value<0.05.

<p>In order to perform hierarchical cluster analysis we constructed a distance matrix using the two-tailed Spearman correlation test, due to the non-normal distribution of expression within samples. The cluster analysis shows a good separation between the two groups of samples, based on the significantly different miRNAs. The microRNA expression data were centered by 2 directions (i.e., by miRNA and patients). Red and yellow represent low and high miRNA expression, respectively.</p

Top-10 most discriminative miRNAs, based on RELIEF score.

<p>Top-10 most discriminative miRNAs, based on RELIEF score.</p

Kaplan-Meier curves for OS and DFS according to (A–B) miR-211 above and below median expression (e.g., low vs. high expression), and to (C–D) k-means clustering.

<p>Kaplan-Meier curves for OS and DFS according to (A–B) miR-211 above and below median expression (e.g., low vs. high expression), and to (C–D) k-means clustering.</p

<i>In vitro</i> studies validating the role of miR-211 in gemcitabine chemosensitivity.

<p>(A) MiR-211 expression in 9 PDAC cell lines and 5 primary tumor cultures. Expression was determined by quantitative PCR, using RNU6 as reference, and the values are in a.u. (B) MiR-211 expression in MIA PaCa-2 and LPc028 cells transfected with negative controls, pre-miR-21 or anti-miR-21 oligos. MiR-211 expression was calculated with the 2^−ΔΔCT method with respect to the expression in cells transfected with negative miRNA controls, set as 1, as represented by the dotted line (C) Gemcitabine antiproliferative effects in MIA PaCa-2 and LPc028 cells transfected with miRNA negative controls, pre-miR-21 or anti-miR-21 oligos. *Significantly different from control transfected cells, which were not treated with gemcitabine, set at 100% (P<0.05). (D) RRM2 mRNA expression in MIA PaCa-2 and LPc028 cells transfected with negative controls, pre-miR-21 or anti-miR-21 oligos. RRM2 expression was calculated with the 2^−ΔΔCT method with respect to the expression in cells transfected with miRNA negative controls, set as 1, as represented by the dotted line. Columns, mean values obtained from three independent experiments; bars, SE. *Significantly different from control (P<0.05).</p