Molecular Mechanism Of CircCpsf6 Regulating Neuronal Growth And Development

Circular RNA(circRNA)is a kind of single-stranded closed and covalent non-coding RNA which is formed by the reverse splicing of pre-mRNA.In recent years,it has been found that circRNAs are widely involved in biological processes such as cell proliferation,differentiation and apoptosis,as well as in the occurrence and development of cardiovascular diseases,diabetes,nervous system diseases and various types of tμmors.They play their biological roles in two main ways.One is to act as molecular sponges of miRNAs,adsorbing miRNAs and regulating the expression level of miRNAs so as to remove the inhibition effect of miRNAs on target genes.The other is to bind proteins,or directly affect the expression level of binding proteins,or affect the downstream signaling pathways associated with proteins.The development of the nervous system is a complex and orderly process,which requires precise regulation of the growth and development of neurons.A large nμmber of non-coding RNAs are involved in the regulation of this process.Many circRNAs are highly expressed in the brain and show tissue-specific and space-time specific.The presence of Circpsf6 was previously identified by circRNA sequencing in mouse and human brain tissues and further verification showed that CircCpsf6 was highly enriched and conserved in the brain.With the extension of neuronal differentiation time,the content of CircCpsf6 gradually increased.Therefore,we hypothesized that CircCpsf6 might play an important regulatory role in the differentiation and development of neurons.However,the exact mechanism is still unclear.Therefore,the main objective of this study is to explore the role and molecular regulatory mechanism of CircCpsf6 in neurodevelopment.In this study,we first selected mouse neuro-like cell line N2a as the cell model of neuronal differentiation.It was found that the content of CircCpsf6 increased with the prolongation of N2a cell differentiation,suggesting that CircCpsf6 may indeed be involved in the differentiation and development of neurons.The formation of neurite is the earliest,basic and critical step in neuronal development.Therefore,we detected the number and length of neurite in mouse N2a cells,human SH-SY5Y cells and primary cultured cortical neurons of mice by immunofluorescence staining.Through the knockdown and overexpression of CircCpsf6,it was proved that CircCpsf6 could promote neurite outgrowth.In order to further explore the molecular mechanism by which CircCpsf6 regulates neuronal development,miRNA sequencing and mRNA sequencing of the CircCpsf6 knockdown model in N2a cells were performed.Through qRT-PCR and dual luciferase assay,it was found that miR-214-5p was the downstream miRNA indirectly regulated by CircCpsf6.The level of miR-214-5p in N2a cells decreased with the extension of differentiation time.MiR-2145p could inhibit neurite outgrowth in N2a cells.The rescue experiment between CircCpsf6 and miR-214-5p showed that the silence of miR-214-5p could rescue the inhibition of neurite outgrowth induced by the knockdown of CircCpsf6.These results suggested that CircCpsf6 could promote neuronal development by regulating the expression of miR-214-5p.Four databases combined with mRNA sequencing data intersected the target genes of miR-214-5p,and Unc5a was verified as the target gene of miR-214-5p through double luciferase assay.The expression level of Unc5a was decreased after the overexpression of miR-214-5p,and the expression level of Unc5a was increased after the knockdown of miR-214-5p.The expression level of Unc5a was also regulated by CircCpsf6.The content of Unc5a was increased with the extension of differentiation time of N2a.Unc5a promoted neurite outgrowth in N2a cells.The restoration experiment between CircCpsf6 and Unc5a showed that the silence of Unc5a reversed the promotion of neurite outgrowth induced by the overexpression of CircCpsf6.These results suggested that CircCpsf6 could promote neuronal development by regulating the miR-214-5p/Unc5a axis.CCK8 was used to test the cell viability in N2a cells and SH-SY5Y cells.It was found that CircCpsf6 could inhibit the cell activity of N2a cells and SH-SY5Y cells.Then,a specific biotin probe was designed at the ring junction of CircCpsf6 to perform the pulldown experiment of CircCpsf6,named RNA antisense purification experiment.At the same time,the proteins bound by CircCpsf6 were pulled down together,and then proteins were eluted and purified.Finally,mass spectrometry detected the proteins,and 385 proteins that may be bound by CircCpsf6 were detected.The possible protein-protein interactions of these 385 proteins were predicted in order to find the powerful proteins.GO analysis and KEGG analysis showed that CircCpsf6 may inhibit the cell viability by affecting cell proliferation or apoptosis through binding proteins.Combined with the differentiation experiment of CircCpsf6 in neurons,we speculated that CircCpsf6 might inhibit the proliferation and promote the differentiation of nerve cells.In summary,our results suggested that CircCpsf6 could promote neuronal development by regulating the miR-214-5p/Unc5a axis,and CircCpsf6 may affect cell viability through binding proteins,thereby regulating neuronal growth and development.The study on the molecular mechanism of CircCpsf6 regulating the growth and development of neurons was of great significance for us to understand the regulatory role of circRNA in the process of neural development,and could also provide some ideas and references for the diagnosis and treatment of related neurological diseases.