Mechanism Of MiR-431 In Mice Deaf Children With Developmental Deformities Study Gene Expression Profiles In The Auditory And Small Ears

Part1Basic Research:Expression profiling-mediated by miR-431in miR-431transgenic mice with hearing defectObjective:miR-431transgenic mice exerts hearing defect as characterized by threshold increased ABR value, suggesting functional role of miR-431in regulating hereditary deafness. Thus, miR-431transgenic mice provides a good animal model to explore the pathogenesis of deafness. In this study, the RNA-Seq approach was performed to identify miR431-miedated genes in the inner ear in order to explore the molecular mechanism of overexpressing miR-431-caused deafness in mice.Method:Extract cochlear total RNA of ABR threshold elevated miR-431transgenic mice and normal ABR threshold wild-type mice for RNA-Seq. By sequencing and bioinformatic analysis of GO annotations, get differentially expressed genes. Then, by cross-screening analysis of miR-431target gene prediction data base and hereditary deafness gene data base, got genetic deafness candidate genes regulated by miR-431. Finally, validated by Real-time RT-PCR method RNA-seq data reliability; through cloning, dual luciferase reporter gene system verification miR-431target genes.Results:1. The ABR threshold value of miR-431transgenic mice are higher than that of the wild-type mice. The ABR threshold of A line Tg mice and Wt mice42.3±14.7dB SPL and27.3±6.9dB SPL, respectively; The ABR threshold of B line Tg and Wt mice were34.8±6.9dB SPL and27.5±4.5dB SPL respectively.2. We identified2344genes which were differentially expressed in the inner ear between miR-431and Wt mice with>1.5fold. Among those differentially expressed genes,921genes were differentially expressed with>2fold. Where for the up-regulated genes,7genes expression were greater than100fold,77genes greater than10fold,627genes greater than2fold. Where for the down-regulated genes,4genes expression were greater than100fold,12genes greater than10fold,194genes greater than2fold.3. By analysis genetic deafness gene mutation data base and RNA-seq results, we identified16differentially expressed genes。There were14genes down regulated in Tg mice, CLDN14、COL11A2、COL2A1、COL4A4、COL9A2、ESRRB、GJB2、MITF、 OTOF、POU4F3、SEMA3E、SIX5、SOX10、USH1C;2genes up regulated and they were GJB3、GRHL2。 4. By analysis genetic deafness gene mutation data base and miR-431target genes predicted data base, we identified eight differentially expressed genes (COL4A4, ESRRB, GJB2, MITF, OTOF, USH1C, SEMA3E and GRHL2) which were potential target genes of miR431and have been reported to correlated to hearing lost in mice and human.5. The expression pattern of eight differentially expressed genes was verified by Real-time RT-PCR and the results were consistent with RNA-seq.6.The dual luciferase reporter genes assay was used to verify that COL4A4and SEMA3E were miR-431target genes.Conclusion:miR-431may functionally regulate hearing loss by targeting COL4A4, SEMA3in mice. Part2Clinical Research:Auditory development after placement of bone-anchored hearing aids soft band among children with bilateral aural atresiaObjective:To evaluate auditory developments of children with congenital bilateral aural atresia after using Bone-anchored hearing aids (Baha) Soft band and to compare them with matched peers with normal hearing.Methods:Sixteen patients with bilateral aural atresia were studied in the Baha Soft band group:6patients were under4years old and the age of the other10patients ranging from4to6years. Twenty-nine children with normal hearing (age ranging from8months to6years) were also studied. Auditory development was assessed at three time intervals: baseline,6months and12months. Infant-Toddler Meaningful Auditory Integration Scale(IT-MAIS) was conducted for children under4years old; Meaningful Auditory Integration Scale(MAIS), Chinese Mandarin lexical neighborhood test(MLNT) and sound field pure tone audiometry(PTA) were used for children of4-6years old.Results:The mean IT-MAIS scores were41±24%,60±22%and73±7%respectively at three time intervals. The mean MAIS scores were66±7%,90±5%, and99±2%. The mean pure-tone threshold of the patients was63±6dB HL unaided and31±4dB HL with Baha Soft band. Mean speech discrimination scores at the three time intervals were74±19%,86±16%, and95±4%with the easy disyllabic list;48±18%,73±15%, and81±7%with the hard disyllabic list;55±17%,74±22%, and83±14%with the easy monosyllabic list; and31±14%,61±15%, and71±13%with the hard monosyllabic list.Conclusion:Baha Soft band is suitable for infants and young children with bilateral aural atresia. The auditory development testings are encouraging and the patients could reach the lower limit of normal hearing.