Molecular and therapeutic mechanisms of differentiation-inducing microRNA miR-506-3p in
neuroblastoma
Abstract
There is a lack of understanding how critical microRNAs (miRNAs) are in controlling neuroblastoma (NBL) cell
differentiation. This prevents the application of miRNA-based therapeutics to NBL differentiation therapy, which
is an approach to induce malignant cells into terminal differentiation and thereby block tumor growth. The long-
term goal of the applicant is to define the role of miRNAs in regulating NBL cell differentiation and make
contributions to the development of miRNA-based differentiation therapy for NBL. The objective of this study
is to elucidate the mechanisms underlying the differentiation-inducing function of a differentiation-inducing
miRNA recently identified in our group, miR-506-3p, and to develop miR-506-3p analogs with enhanced
differentiation-inducing activity. The central hypothesis is that miR-506-3p functions as a inducer of cell
differentiation through directly targeting a group of genes that play key roles in regulating NBL cell
differentiation, and that miR-506-3 analogs with enhanced differentiation-inducing activity can be developed by
modifying the nucleotide sequence in the non-seed region of the wildtype miR-506-3p. This hypothesis is
supported by strong preliminary data generated in the applicant's lab. The following Specific Aims are
proposed: Aim 1, Identify novel miR-506-3p targets that mediate its differentiation-inducing function. A
functional high-content screening (HCS) approach will be used to systematically investigate its targets
regarding their role in regulating NBL cell differentiation. The direct interactions of miR-506-3p with the targets
identified from screen will be validated by combining a biotinylated-miRNA pull-down assay and a luciferase
reporter assay. Since the molecular mechanisms of regulating NBL cell differentiation are still poorly
understood, we expect that a comprehensive investigation of the miR-506-3p targets will reveal genes that
were previously unknown to regulate NBL differentiation. Aim 2, Develop novel miR-506-3p analogs with
enhanced differentiation-inducing activity. Synthetic analogs of miR-506-3p will be designed, and analogs with
significantly increased differentiation-inducing activity relative to wildtype miR-506-3p mimic will be identified
using HCS and further in vitro validation analysis. The identified analogs will then be preliminarily evaluated for
their therapeutic potential by examining their generic differentiation-inducing activity in a panel of NBL cell lines
with diverse genetic background, and by examining their effect on viability of non-NBL cells in order to select
analogs with minimum non-specific cytotoxicity. The study is innovative because it will elucidate a novel cell
differentiation pathway in NBL mediated by miR-506-3p and its target genes, and it will identify novel miRNA-
506-3p analogs that has potential to be developed as differentiation therapeutic agents. The study is
significant because it is expected to advance the understanding on the mechanisms of NBL cell
differentiation, and to pave the way to develop more effective miRNA-based differentiation therapies for
treating NBL, which is expected to eventually benefit the survival and wellness of NBL patients.