MicroRNA‐506‐3p as a Differentiation Agent for Neuroblastoma
Authors: Sousares Michaela , Du Liqin 
Published: November 2023

Background

Neuroblastoma (NB) is the most common solid tumor cancer in infants less than 1 year of age. About 60% of the newly diagnosed cases have already spread to the lymph nodes, where children with this disease typically don't live past 10 years of age. NB arises from the failure of neural crest cell precursors to differentiate, which inhibits the maturation of these cells, leaving immortal, infant, malignant cells. The identification of new targets involved in differentiation is crucial for the elucidation of this disease. Differentiation therapy involves the induction of malignant cells to mature and undergo apoptosis, and relies on the discovery of targetable biological molecules that play a role in NB cell differentiation.

MicroRNAs (miRNAs) are ~20 nucleotide long, noncoding RNAs, which have been determined to regulate transcription and the expression of many genes within the cell. MiRNAs have been used as tumor suppressors to inhibit tumor growth. MiRNA mimics are partially double‐stranded RNAs designed to mimic the function of endogenous miRNAs, therefore increasing the miRNA levels in the cell.

Using a high‐content screening technique to visualize neurite outgrowth of NB cells, Du and group identified 14 novel miRNAs that induce differentiation. MiR‐506‐3p was identified as the most potent miRNA and is seen highly regulated in NB cells, which may be the primary driving force in controlling cell differentiation. MiR‐506‐3p also showed a more potent effect than 13‐cis‐retinoic acid, suggesting this as an effective differentiation agent for neuroblastomal remission therapy.

The goal of this study is (1) to determine the NB cell sensitivity to miR‐506‐3p mimic‐induced cell death in NB cell lines with different genetic backgrounds, and to determine the correlation between the sensitivity to All Trans Retinoic Acid (ATRA) and miR‐506‐3p mimic; (2) to determine the correlation of cell sensitivity to these treatments with genetic alterations, such as MYCN amplification; (3) to determine whether the combined treatment of miR‐506‐3p mimic and retinoic acid will have a synergistic or additive effect on cell viability.

Methods

To carry out these aims, the NB cells are treated with ATRA and the miR‐506‐3p mimic, and then are analyzed for neurite outgrowth, or maturation of the cells. Cell viability is determined with an MTT assay. After treatment with ATRA, the confirmation of differentiation is detected using Western Blot, with known NB differentiation markers (i.e. growth associated protein 43, β Tubulin (III), neuron specific enolase, and Calnexin).

Expected Results

It is anticipated that following treatment with ATRA and the miR mimic, the NB cells will exhibit a strict correlation and cell sensitivity to these treatments. The NB cells with different genetic backgrounds, such as MYCN amplification, may display a decreased sensitivity to ATRA and the miR mimic, while combined treatment may provide a synergistic effect on the NB cells.

Conclusions

If a strict correlation between ATRA and the miR mimic is observed, this may be indicative of the same pathway being targeted by these treatments, allowing for more insight into the mechanisms of differentiation loss in NB cells. After observing a synergistic effect from combined treatment of ATRA and miR‐506‐3p mimic on NB cells, it may be determined that different treatment methods may be more effective than traditional methods, in treating patients (i.e. high‐risk) with this disease.

Support or Funding Information

DoD PRMRP Discovery Award (PR151532) 09/01/2013‐02/28/2017

Goal: To comprehensively identify neuroblastoma differentiation‐inducing microRNAs by combining a functional high‐content screening approach and a library of microRNA mimics used to raise the intracellular microRNAs levels.

Startup funds from Texas State University. 09/01/2016‐present.

Goal: To investigate the mechanisms of neuroblastoma cell differentiation.