MicroRNA-124 Regulates Neurite Outgrowth By Targeting Retinoic Acid Receptor Gamma

During the development of central nerve system in mammals,neurite outgrowth is required for a series of neural activities such as brain development,neuroregeneration and neural path and the establishment of synaptic connection and so on.It is of great significance to focus on the study of neurite outgrowth in the research on the treatment of neurodevelopmental diseases,neurotraumatic diseases and neurodegenerative diseases.Therefore,it is important to understand the molecular mechanisms that regulate neurite outgrowth to find more effective drugs of neurological diseases.All-trans retinoic acid(RA),as a metabolite of vitamin A,is an important exogenous supplemental nutritional factor in the growth and development of various cells.It is also widely used as a factor to induce neurite outgrowth.Retinoic acid receptors are widely expressed in the central and peripheral nervous systems of mammals.The role of retinoic acid receptor gamma(RARG)-one of the retinoic acid receptors in neurite outgrowth is not well understood.MicroRNA-124(miR-124),a neuron-specific microRNA,has been implicated in promoting neurite growth and simultaneously exhibiting increased expression level during neural differentiation.However,little is known about the relationship between miR-124 and retinoic acid signaling system in neurite outgrowth.Mouse Neuroblastoma cells(N2a cells),P19 embryonal carcinoma cells(P19 cells),and mouse primary cortical neurons were applied to investigate the roles and the underlying molecular mechanisms of miR-124 and RARG on neurite outgrowth.Respectively,molecular biology technology,cell culture,immunoblotting,and cell morphological methods were performed for this study.Here,we showed that RARG exhibited decreased expression,whereas miR-124 exhibited increased expression during neuronal differentiation in N2a cells,P19 cells,and mouse primary cortical neurons,as detected by immunoblotting or RT-qPCR.And we found that miR-124 inhibits RARG expression by binding to the 3'UTR(3'untranslated region)of RARG through a luciferase reporter assay.Moreover,upregulation of miR-124(using miR-124 overexpressing plasmid or miR-124 mimic)resulted in a significant decrease in RARG protein in N2a cells and neurons.Therefore,we proposed the hypothesis that whether and how the miR-124/RARG axis regulates neuronal outgrowth,which is not well understood.In our study,RARG knockdown by shRNA promotes neurite growth,whereas RARG overexpression(coding domain sequence,without 3'UTR)represses neurite growth in three cell models we used.Furthermore,RARG overexpression could reverse the enhancing effect of the upregulation of miR-124 on neurite outgrowth,and RARG knockdown could partially rescue neurite outgrowth defects caused by miR-124 inhibitor.Collectively,the data reveal that miR-124/RARG axis is crucial for neurite outgrowth.RARG is proved for the first time as a new target regulated by miR-124 and it regulates neurite outgrowth.This provides further clues for finally revealing the precise molecular mechanisms in neural development.