Pcolce2 And Rps14 Regulate The Regeneration Of Inner Ear Hair Cell By AAV

Hearing loss,as the third leading cause of disability in the world and the first among sensory disorders,is a global health problem that needs to be resolved urgently.Among them,the main causes of sensorineural hearing loss（SNLS）include damage to hair cells（hair cells,HC）and neurons in the inner ear.HC in the inner ear acts as a sound receptor on the auditory epithelium,it can convert the received physical signal of sound waves into a chemical signal.Early studies have demonstrated that mammalian HCs cannot regenerate in adulthood,but Lgr5 positive supporting cells（Lgr5⁺SCs）have been found to have the characteristics of inner ear stem cells and can regenerate HCs under specific injury conditions.At the same time,the regulation of Atoh1,Gfi1,Pou4f3,and other genes,as well as Wnt,Notch,and other signaling pathways can induce SCs to regenerate into HCs,which provides new treatment ideas for HC regeneration.In recent years,research has achieved certain results in regenerating HCs through gene regulation.However,there are considerable differences in morphology and function between regenerated HCs and the native HCs.Therefore,it is of great significance and value to explore the genes and mechanisms related to HC regeneration.The background of the previous research was that by comparing damaged and undamaged Lgr5⁺progenitors by neomycin,Lgr5⁺progenitors and Lgr5^-supporting cells,and Lgr5⁺progenitors from the apical and basal of the cochlear with the RNS-seq analysis.By comparing the common differentially expressed genes of these three groups,it was found that 37 differential genes deserve further study.Among them,the differential genes Pcolce2 and Rps14 were shown to be related to the proliferation of inner ear stem cells in previous experiments,so this dissertation focuses on Pcolce2 and Rps14 to explore their possible role in HC regeneration.Pcolce2（procollagen C-endopeptidase enhancer 2）encodes a glycoprotein,is a relevant biomarker for the clinical prognosis of many cancers,and is screened here as a candidate regulator of SC plasticity.At the same time,Rps14（ribosomal protein S14）,also known as ribosomal protein S14,is a structural protein of the 40S ribosomal subunit involved in ribosome biogenesis.It was explored as a candidate gene promoting SC proliferation and differentiation in this study.This study used an optimized AAV-mediated gene overexpression system（a newly developed AAV for efficient transfection of inner ear supporting cells,called AAV-ie）to achieve overexpression of target genes in mouse cochlear SCs.First,through immunohistochemistry and western blotting,it was found that PCOLCE2 was mainly expressed and localized in HC in the mice cochlea of different ages.Subsequently,through 2D and 3D culture experiments of inner ear stem cell organoids in vitro,it was found that overexpression of Pcolce2 significantly promoted the proliferation of organoid solid spheres;in young WT mice,AAV was injected through the cochlear round window to achieve overexpression of Pcolce2 in SCs.The proliferation of SCs was detected through Ed U labeling,and there is a tendency to differentiate into HCs to a certain extent.At the same time,q PCR was used to confirm that overexpression of Pcolce2 can activate downstream pathways related to HC formation at the transcriptional level.In stem cell differentiation experiments,in vivo and in vitro experimental results showed that overexpression of Pcolce2 did not affect the differentiation function of normal mouse HC,and the results of lineage tracing also reached the same conclusion.However,in a mouse model of hair cell injury,it was found that Pcolce2 can promote the differentiation of HCs after injury and further promote HC regeneration after injury under the combined action of Wnt agonists and Notch inhibitors.Through staining and lineage tracing,it was confirmed that regenerated HCs are mainly derived from SCs.Finally,transcriptome analysis of regenerated HCs combined with related q PCR verification found that Pcolce2promotes HC regeneration mainly through the Wnt classic pathway,which is mainly manifested by the up-regulation of wnt10a,lgr5 and other genes,and the down-regulation of the proliferation-related gene p27^kip1.Another gene investigated in this study,Rps14,is a gene related to ribosome production.According to previous studies,this gene is mainly expressed in HCs and SCs.Organoid culture results show that Rps14 can promote the proliferation of inner ear stem cells under 2D and 3D culture conditions,doubling the number of organoids.It can also improve the stemness of organoids,with stemness-related markers increasing by more than 2.5 times during two-dimensional culture.At the same time,Rps14 can also increase the number and proportion of HCs derived from stem cell differentiation.In in vivo experiments,AAV-Rps14 was delivered into Sox9^{Cre ER}/Rosa26-td Tomato^loxp/mice through the cochlear round window.Through lineage tracing,it was found that AAV-Rps14 can promote the regeneration of Sox9⁺SCs into HCs in vivo.At the same time,after neomycin injury,overexpression of Rps14 will also promote the increase of SC-derived HCs.These results provide a reference for research on the mechanism of gene regulation of inner ear HC regeneration.In summary,this dissertation explores the functions of Pcolce2 and Rps14 in HC regeneration and confirms that both of them play an important role in promoting HC regeneration and have the potential to become regenerative targets for the treatment of hearing loss caused by HC damage.It provides theoretical guidance to promote HC regeneration and restore damaged auditory function.