Background: We first identified CHD5 as a tumor suppressor gene that is frequently deleted in NBs. Mutation of the remaining CHD5 allele is rare in these tumors, yet expression is very low or absent, so expression is likely regulated by epigenetic mechanisms. MicroRNAs (miRNAs) are small RNAs that bind to the 3’UTR to mediate downregulation of target gene expression by translational repression or cleavage of the target genes’ mRNA. In order to understand the role of miRNA regulation of CHD5 in NB cell lines we tested miRNAs predicted to target CHD5.
Method/approach: We used bioinformatic programs and identified 18 miRNAs that were predicted to bind to the CHD5-3’UTR. We used a renilla-luciferase reporter plasmid that contains 103 bp of the 3’UTR of CHD5 targeted by miRNAs. We performed transient transfections in NLF and SY5Y NB cell lines with the reporter plasmid and miRNA mimic. We also used control plasmids and Allstar siRNA as another negative control. Western blot analysis was performed using whole cell extracts of NBLS to further validate the functional regulation of CHD5 expression by miRNAs.
Results/Conclusion: We found seven miRNAs that significantly downregulated CHD5 expression in NBs: miR-211, 17, -93, -20b, -106b, -204, and -3666. Interestingly, MYCN upregulates four of the candidates we identified: miR-17, -93, -106b & -20b. This suggests that miRNAs driven by MYCN and other genes represent a potential epigenetic mechanism to regulate CHD5 expression. Our western results indicate there was almost complete reduction of CHD5 protein levels in NBLS cells transfected with miR-211, miR-17, miR-93 and miR-20b, but no changes were observed in CHD4, actin or MYCN protein levels. These results strongly suggest that miR-211, -17, -93 and -20b can dramatically downregulate CHD5 protein expression in NBs, and MYCN amplification and over expression can contribute to this.