Construction Of CeRNA Network And Study On The Synaptic Regulation Of Inner Ear Hair Cells

BackgroundSensorineural hearing loss(SNHL)accounts for a large part of the hearing loss,which is mainly caused by the loss of sensory hair cells or the impairment of the afferent nerve to the auditory cortex.The abnormal signal transmission between hair cells and spiral ganglion cells caused by the change in the number of synapses or dysfunction in the inner ear is one of the important causes of sensorineural deafness.Genetic mutations and environmental factors can cause irreversible damage to hair cells or lead to degeneration of synapses in inner hair cells,leading to hearing loss."Cochlear synaptopathy"(CS)refers to the loss of synaptic transmitters between hair cells and auditory nerve fibers in the inner ear when the number of hair cells remains unchanged,which may be one of the important pathogenesis of SNHL,especially presbycusis.Previous studies by our group have found that Dynamin1 is a key protein for maintaining synaptic vesicle enestion and is regulated by mi RNA-30 b and miRNA-140.Meanwhile,we found that Dynamin1 and mi R-30b/miR-140 were differentially expressed in the inner ear at different ages,that is,the expression of Dynamin1 decreased in the elderly mice,while the expression of miR-30 b and miR-140 was up-regulated in the elderly mice.but the deeper regulatory mechanism of Dynamin protein and other genes involved in synaptic regulation are still unknown.LncRNA can adsorb some specific miRNAs by means of bait and regulate the expression of these miRNA target genes,this mode of action is called "miRNA sponge",and lncRNA with this function is called competitive endogenous RNA(ceRNA).It has been shown to affect the development of many diseases.Therefore,1.Reveal the changing relationship of the whole transcriptome with the increase of age,and construct the potential ceRNA network of lncRNA,miRNA-140 / miRNA-30 b,Dnm1.2.By analyzing differentially expressed genes,we can identify other synaptic regulatory genes except Dnm1 with age,which is of great significance to the study of the pathogenesis of synaptic injury.Methods1.We conducted transcriptome sequencing on the basal membrane of C57BL/6J mice at 4 weeks(adult)and 40 weeks(elderly),and looked for differential expressions of lncRNA(DELncRNA),mRNA(DEmRNA)and mi RNA(DEmiRNA).The endogenous competitive inhibitory RNA(ceRNA)network of lncRNA-miRNA-mRNA was constructed by miRanda and targetscan.2.Genes differentially expressed in different age groups(FC > 1.5,P < 0.05)were analyzed,and the main functions of differentially expressed genes were explored through pathway enrichment analysis using gene ontology(GO)database and KEGG database.Screening for synaptic regulatory differential genes.3.Quantitative real-time PCR and immunofluorescence staining were used to verify the differences of synaptic regulatory genes.Results1.A total of 3,267 DEGs were screened,among which 1521 DEGs were down-regulated and 1746 DEGs were up-regulated.There were a total of 1205 DELncRNA,of which 537 were up-regulated and 668 were down-regulated.A total of 303 DEmiRNA were identified,of which 134 were up-regulated and 169 were down-regulated.RNA with ceRNA regulatory relationship with miR-30b/miR-140,Dnm1 and correlation coefficient >0.9 are:Gm10684,2900079G21 Rik,1700036A12Rik,B830012L14 Rik,Gm11815,Gm19967?2.Functional enrichment of the differentially expressed genes showed that the down-regulated genes were mainly involved in voltage gated ion channel activity regulation and synaptic regulation,while the up-regulated genes were mainly involved in immune regulation.A total of 64 synaptic regulatory genes were screened,among which 6 potassium channel genes(Kcnq2,Kcnk3,Kcnmb4,Kcnk9,Kcnh5 and Kcnc2)showed the highest enrichment.3.qRT-PCR verified the differential expression of 6 genes regulating potassium channels of synapses in the inner ear in different age groups.Immunofluorescence showed that the expression of KCNQ2 and KCNMB4 decreased significantly in the inner ear of aged mice.Conclusion1.This study provided the basic data of transcriptome expression in the basement membrane of the inner ear of aged mice,identified DELncRNA,DEmRNA and DEmiRNA,and integrated their potential ceRNA networks.RNA with ceRNA regulatory relationship with miR-30b/miR-140,Dnm1 and correlation coefficient of > 0.9 are:Gm10684,2900079G21 Rik,1700036A12Rik,B830012L14 Rik,Gm11815,Gm19967?This will lay a foundation for further study on the regulatory mechanism of Dynamin1.2.Synaptic regulatory genes were screened,and the expression of potassium channel genes(Kcnq2,Kcnk3,Kcnmb4,Kcnk9,Kcnh5 and Kcnc2)decreased significantly.It is suggested that it may play an important role in the change of synapse function in presbycusis,which will help us to better understand the mechanism of the occurrence of synapse damage in presbycusis,and provide new ideas for the prevention and treatment of presbycusis in the future.