Study On The Implication Of MicroRNA 384 Targeting Notch Signal Pathway For The Regeneration Of Impaired Hair Cells

Object: Impaired hair cells in non-mammals were found to be reproducible while those in mammalian cochlea were not.However,it has been reported that hair cells in mammalian vestibular are able to regenerate in a limited extent after injury.Notch pathway plays an essential role in the regeneration of hair cells.Lateral inhibition induced by Notch signal pathway could suppress the expression of ATOH1(a crucial factor of hair cells generation and development)and prevent the differentiation of supporting cells into hair cells.It was noteworthy that the inhibition of Notch signal pathway and up-regulated ATOH1 was observed in the regeneration of hair cells in mammalian vestibular.However,the mechanisms of Notch signal pathway implicated in regulating the regeneration of hair cells still remained unclear.MircoRNAs(miRNAs),targeting Notch signal pathway,could be the important regulatory factor functioning in RNA silencing and post-transcriptional regulation.This study is meant to filter miRNAs regulating Notch pathway in inner ear,and unravel its influence to the regeneration of impaired hair cells in vitro.Method: literature was entensively refered in order to gather differential-expressed miRNAs in inner ear.Databases,for instance KEGG Pathway,String and targetscan,were retrieved to get candidated or predicted interaction between Notch pathway related genes and miRNAs.A biological interaction network was constructed based on this.Topological analysis was executed to find the core gene in this network and the subnetwork Core-Notch was extracted.Less studied miRNAs which was predicted to regulate Notch pathway was obtained after displaying topological analyse and GO analyse to Core-Notch network.Several tissues include cochlea were detached and Q-PCR was performed to detect differential expression of miRNA and target gene.MiRNAs and target genes binding sites were predicted with targetscan and 3'UTR-WT-psiCHECK2 vectors of target genes were constructed.Then 3'UTR-MUT-psiCHECK2 vectors were attained by point mutation PCR.MiRNA mimic and wild type or mutant vector were transfected into 3T3-L1 cells.Dual luciferase reporter gene assay was displayed to validate regulation of miRNAs to target gene.Result: In this study,the interaction network,including key genes in Notch signal pathway and differential-expressed miRNAs in inner ear,was constructed by bioinformatics methods.Subnetwork,Core-Notch,was extracted based core genes filitered.Go analysis of Core-Notch reveals its reliability as its enrichment in Notch pathway as well as inner ear receptor cell fate commitment.And then,miRNA-384 was identified via topological analysis and GO analysis,which could down-regulate Notch1 and Dll4,core genes in Notch pathway.The subsequent experimental results showed that the expression of microRNA-384 is specifically expressed in brain and inner ear and the expression of Notch1 is significantly down-regulated in miRNA-384-mimic-transfected HeLa cells.The transfected expression vectors with wild or mutant miRNA-384 binding site(Notch1(Dll4)-3'UTR–WT-psiCHECK2)and(Notch1(Dll4)-3'UTR-MUT-psiCHECK),as well as miRNA-384 mimic,were co-transfected in to HEK293 T cells,and then the dual-luciferase reporter gene assay was conducted and validate the effect of miRNA-384 on the down-regulation of Notch1.Conclusion: MiRNA-384 expressed specially in inner ear is able to target Notch1 and Dll4,and may play a potential role in the the regeneration of iImpaired hair cells.