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.
The goals of this project are to elucidate the mechanisms underlying the differentiation-inducing function of miR-506-3p and develop miR-506-3p analogs that have high potential to be used as differentiation therapeutic agents for treating neuroblastoma. The proposed research is relevant to public health because the further successful development of the identified analogs into effective therapies for neuroblastoma will result in survival benefit for neuroblastoma patients. Thus, the proposed investigation is consistent with the NCI mission of fighting cancer and is relevant to the NIH mission to acquire and apply knowledge to reduce the burden of illness and disability.
