Classifying Medulloblastoma Subgroups Based on Small, Clinically Achievable Gene Sets

As treatment protocols for medulloblastoma (MB) are becoming subgroup-specific, means for reliably distinguishing between its subgroups are a timely need. Currently available methods include immunohistochemical stains, which are subjective and often inconclusive, and molecular techniques—e.g., NanoString, microarrays, or DNA methylation assays—which are time-consuming, expensive and not widely available. Quantitative PCR (qPCR) provides a good alternative for these methods, but the current NanoString panel which includes 22 genes is impractical for qPCR. Here, we applied machine-learning–based classifiers to extract reliable, concise gene sets for distinguishing between the four MB subgroups, and we compared the accuracy of these gene sets to that of the known NanoString 22-gene set. We validated our results using an independent microarray-based dataset of 92 samples of all four subgroups. In addition, we performed a qPCR validation on a cohort of 18 patients diagnosed with SHH, Group 3 and Group 4 MB. We found that the 22-gene set can be reduced to only six genes (IMPG2, NPR3, KHDRBS2, RBM24, WIF1, and EMX2) without compromising accuracy. The identified gene set is sufficiently small to make a qPCR-based MB subgroup classification easily accessible to clinicians, even in developing, poorly equipped countries

MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression

3′UTR of Dicer 1 is directly targeted by miR-192.

<div><p>The dual luciferase assay detected that Dicer1 is modulated by miR-192 in NB cell lines. The relative luciferase unit (RLU) was measured in SHEP (A) or NUB (B) cells.</p> <p>A. The dual-luciferase assay resulted in a significant reduction of RLU of WT Dicer1 (3' UTR of Dicer1 wild type) following transfection with miR-192-vec (*p=0.049).</p> <p>B. Following transfection with miR-192 mimic, WT Dicer1 RLU was significantly decreased (*p=0.0003). Following mutagenesis, cells were transfected with Dicer1 plasmid in which mutations were introduced in all three BSs of Dicer1 (MUT ALL); active BS1 (mutated at BS2+BS3)(* p=0.004); active BS2 (mutated at BS1+BS3) (* p=0.04) and active BS3 (mutated at BS1+BS2). </p> <p>Values are expressed as the mean ± SE of combined results from three independent experiments. </p></div

Site-directed mutagenesis of miR-192 binding sites on 3' UTR of Dicer1.

<div><p>Three mutations were introduced into the three potential binding sites of miR-192 on 3' UTR of pI DICER1 using Multi Site-Drected Mutagenesis Kit .</p> <p>The nucleotides that were mutated are circled and were changed to the nucleotides in bold. </p></div

Kaplan Meier analysis by miR-192 expression.

<p>Kaplan Meier analysis for PFS by miR-192 expression: high and low miR-192 expression levels were determined as above or below the median expression level and were analyzed in (A) a whole cohort (n=43) and in (B) a cohort following exclusion of MYCNA (n= 36) .</p

miR-192 is associated with NB cell viability, proliferation and migration capability.

<div><p>Following transfection of miR-192 mimic and inhibitor, we measured the viability of NUB6 cells by direct counting (A), proliferation properties of the cells by XTT analysis (B) and the migration ability of NUB6 cells (C).</p> <p>A. Inhibition of miR-192 resulted in significantly decreased cell viability (*p=0.042).</p> <p>B. Following transfection of miR-192 mimic and inhibitor, a significant increase(*p=0.044) or decrease (*p=0.038) was detected in the proliferation rate respectively.</p> <p>C. Overexpressing miR-192 resulted in a significant increase in cell migration (*p= 0.00036). </p> <p>Values are expressed as the mean ± SD of combined results of three independent experiments.</p></div

miR-192 regulates Dicer1 mRNA expression.

<div><p>Following transfection of miR-192 mimic and inhibitor, a significant decrease (*p=0.028) or elevated Dicer1 mRNA expression levels were detected, </p> <p>respectively.</p></div

Kaplan Meier analysis by Dicer1 expression.

<p>Kaplan Meier analysis for PFS by Dicer1 expression (n=47): high and low expression levels of Dicer1 were determined as above (n=24) or below (n=23) the median expression level.</p

miR-192 regulates Dicer1 protein expression.

<div><p>A. Dicer1 protein expression in NUB6 cells, as evaluated by Western blotting, after exposure to miR-192.</p> <p>B. Graphic illustration of relative Dicer1 protein levels (normalized to tubulin) in Nub6 cells following transfection with miR-192 mimic (*p=0.04) and inhibitor.</p> <p>Data are presented as means ± SE of three independent experiments.</p></div