A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells.

Zhenze Zhao,Monica Li,Alexander Pertsemlidis,Derek C Sung,Yidong Chen,Maggie Zhang,Xiuye Ma,Xiaojie Yu,Liqin Du,Spencer D Shelton,Michael D Losiewicz,Yuanhang Liu,Daniel Hernandez

doi:10.18632/oncotarget.12676

Abstract

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

Highlights

MYCN amplification occurs in about 20% of neuroblastoma cases and correlates with advanced-stage disease and poor patient outcomes [1,2,3,4]
In order to examine the role of differentiationinducing miRNAs in regulating MYCN expression in neuroblastoma cells, we overexpressed a group of thirteen differentiation-inducing miRNAs that we identified previously [14] using miRNA mimics, synthetic oligonucleotides used to raise intracellular miRNA levels, in a neuroblastoma cell line BE(2)-C, the cell line that we used to identify the differentiationinducing miRNAs through high-content screening [14]
We further examined the effect of the thirteen miRNAs on MYCN expression at the mRNA level

Summary

INTRODUCTION

MYCN amplification occurs in about 20% of neuroblastoma cases and correlates with advanced-stage disease and poor patient outcomes [1,2,3,4]. Its role in neuroblastoma tumorigenesis is not fully understood, studies have shown that N-Myc likely fulfills its oncogenic function through simultaneously stimulating expression of multiple oncogenic pathways and repressing expression of multiple tumor suppressive pathways [6, 7], and that inhibiting the differentiation of neuroblastoma cells is one of the important molecular mechanisms underlying its oncogenic function [8,9,10]. Recent studies suggest that microRNAs (miRNAs), a class of endogenously expressed, small non-coding RNAs that regulate gene expression at the translational level, play an important role in the MYCN-mediated oncogenic pathway [7]. We investigate whether the differentiation-inducing miRNAs regulated MYCN expression, whether N-Myc controls the expression of these miRNAs, and we further investigated whether N-Myc plays a role in mediating the differentiationinducing functions of the miRNAs

RESULTS

DISCUSSION

MATERIALS AND METHODS