MicroRNA dysregulation in response to RAR beta 2 inhibition reveals a negative feedback loop between MicroRNAs 1, 133a, and RAR beta 2 during tail and spinal cord regeneration in the adult newt

Background: The molecular events underlying epimorphic regeneration of the adult urodele amphibian tail and caudal spinal cord are undetermined. Given the dynamic nature of gene expression control by retinoic acid (RA) signaling and the pleiotropic effects of microRNAs (miRNAs) on multiple mRNA targets in this complex system, we examined whether RA signaling through a specific receptor, RAR beta 2, alters expression of select miRNAs during spinal cord regeneration. Results: An initial screen identified 18 highly conserved miRNAs dysregulated in regenerating tail and spinal cord tissues after inhibition of RAR beta 2 signaling with a selective antagonist, LE135. miRNAs let-7c, miR-1, and miR-223 were expressed within the ependymoglial cells, coincident spatially with the expression of RAR beta 2. Altering the expression pattern of these three miRNAs led to a significant inhibition of caudal ependymal tube outgrowth by 21 days post tail amputation. We demonstrated that miR-1 targets the 3'-untranslated region of RAR beta 2 mRNA in vitro; and in vivo, up-regulation of miR-1 led to a significant decrease in RAR beta 2 protein. Conclusions: These and previous data suggest that miR-1 and miR-133a, both members of the same miRNA gene cluster, may participate with RAR beta 2 in a negative feedback loop contributing to the regulation of the ependymal response after tail amputation. Developmental Dynamics 244:1519-1537, 2015. Key Findings * This represents the first evidence of microRNAs regulated by retinoic acid signaling in a regeneration competent organism. * In vivo alterations in the expression of any of microRNAs let-7c, miR-1 and miR-223 inhibits tail and spinal cord regeneration. * miR-1 and miR-133a are associated with a negative feedback loop with retinoic acid receptor beta in ependymoglial and subependymoglial cells in the regenerating newt spinal cord.