CircRtn4 Acts As The Sponge Of Mir-24-3p To Promote Neurite Growth By Regulating CHD5

Billions of specific neurons build the neural circuits,some of which underlie animal behaviors including cognition,learning and feeling.Neuronal differentiation is a critical step in wiring neural circuits to establish precise neural connections for normal physiological function.Impaired development of neural circuits was associated with behavioral,cognitive and mental disorders,such as epilepsy,intellectual disability,autism,schizophrenia,etc..Therefore,it is important to elucidate the molecular mechanisms underlying the development of neurons.Circular RNAs(circRNAs)are a class of noncoding RNAs derived from back-splicing of pre-mRNA.CircRNAs are characterized as covalently closed single-stranded RNA molecules without a 5' cap and a 3' polyadenylated tail.In recent years,increasing studies have shown that many circRNAs are enriched in the brain,especially in neurons,which suggests that circRNA may play an important role in the nervous system.It was reported that significantly increased expression of mmu_circ₀000250(circRtn4)is associated with the differentiation of primary neurons.In our study,we explored the role of circRtn4 in neurite growth and the molecular mechanism.In this study,we confirmed that the expression level of circRtn4 increased significantly after the differentiation of N2a cells and primary cortical neurons.Subsequently,circrtn4 were knocked down in N2a cells and primary cortical neurons by transfecting siRNA.And then we found that length of the longest neurite and total length of neurites of N2a cells were significantly decreased,while their branch numbers showed no change.Cortical neurons showed similar effects on their longest neurite length and total neurites length,while their branch numbers were reduced.Otherwise,both N2a cells and cortical neurons overexpressing circRtn4 exhibited increased branch numbers,increased length of the longest neurite and increased total length of neurites.These results indicate that circRtn4 promote neurite growth.Fluorescence in situ hybridization(FISH)and nuclear cytoplasmic separation experiments results confirmed that circRtn4 mainly located in the cytoplasm,where circRNAs often function as miRNA sponges.Then,we screened 12 miRNAs candidates predicted by miRDB.Dual-luciferase reporter assay and RNA antisense purification assay(RAP)revealed that miR-24-3p can interact with circRtn4 directly.Corresponding to the increased expression of circRtn4,the expression level of miR-24-3p decreased observably with the differentiation of N2a cells and primary cortical neurons.To explore the function of miR-24-3p in neurite growth,miR-24-3p mimic or miR-24-3p inhibitor was transfected into N2a cells and cortical neurons.The data showed that miR-24-3p could inhibit neurite growth.In addition,we mutated the binding site of miR-24-3p on circRtn4 overexpression plasmid.Subsequently,circRtn4 mutant form Mut-circRtn4 were overexpressed in cortical neurons and N2a cells.The promoting effect of circRtn4 on neurite growth did not exist in neither cortical neurons nor N2a cells overexpressing Mut-circRtn4,which indicated that circRtn4 promotes neurite growth at least partly by miR-24-3p.In order to explore the downstream target genes of circRtn4/miR-24-3p,transcriptome RNA sequencing was employed to identify differential genes after circRtn4 knockdown in N2a cells.The down regulated genes were crossed with the target genes of miR-24-3p predicted by the database.There were six possible target genes.By dual luciferase reporter assay,we found that CHD5 can interact with miR-24-3p directly.And Western Blot showed that the protein level of CHD5 was regulated by circRtn4 and miR-24-3p.Similar to circRtn4,the differentiation of N2a cells and primary cortical neurons increased the expression level of CHD5.Then rescue experiment was designed and we found that knockdown of CHD5 was enough to reverse the promoting role of circRtn4 overexpression in neuronal differentiation.In conclusion,our study showed that circRtn4 can promote neurite growth.And it regulates neurite growth by sponging miR-24-3p to enhance CHD5.