CpG Site Hypermethylation Of The Promoter Region Of Connexin26Gene In The Inner Ear Of Mimetic Aging Rats Induced By D-galactose

PartⅠ Establishment of D-galactose induced inner ear aging rat model with mtDNA4834bp deletionObjective:To establish the mimetic aging of inner ear rat model with mtDNA common deletion induced by D-galactose.Method:48wistar rats were divided randomly into2groups:D-gal group(500mg/kg/D) were treated with subcutaneous injection for8weeks; control group(saline group) were given saline of equal volume for8weeks. The hearing level were tested by auditory brainstem response(ABR). The mitochondrial DNA4834bp deletion was tested by Real-time PCR.Result:The difference in shift of hearing threshold among the two group was not significant(P>0.05).The rate of mtDNA4834bp deletion in D-gal group was higher than that in the control group, there was a significant difference between the two groups(P<0.05).Conclusion:We have established the model of mimetic aging rat model with mtDNA4834bp deletion successfully. PartⅡ CpG site hypermethylation of the promoter region of connexin26gene in the inner ear of mimetic aging rats induced by D-galactoseObjective:To explore the expression level and the aberrant methylation of the promoter region of connexin26gene in the cochlea of inner ear aging rats.Method:The RT-PCR and Western Blot were used to detected the expression of connexin26on the mRNA and protein levels. The methylation status of promoter region of GJB2was detected by bisulfite sequencing analysis.Result:we applied a mimetic aging rat model by D-gal injection for8weeks and demonstrated that D-gal injection for8weeks can increase the common deletion (CD) incidence in the inner ear of rat without affecting the Auditory Brainstem Response (ABR). Western blot of rat inner ear tissue indicated that D-gal injection decrease the protein level of Connexin26(Cx26), one of the major protein subunits forming Gap Junctions (GJs) which are important in maintaining homeostasis in the inner ear and normal hearing. Consistent with this result, Real-time RT-PCR showed a similar decrease of the mRNA level of Cx26. Further bisulfite sequencing analysis revealed that D-gal injection can induce hypermethylation of the CpG site of the promoter region of Cx26gene in the inner ear tissue.Conclusion:Our data indicate that D-gal injection for8weeks can induce CpG site hypermethylation of the promoter region of Cx26gene, which consequently affects the mRNA level of Cx26and leads to the decrease of Cx26protein level. Given the important role of Cx26in the inner ear tissue, we conclude that aberrant DNA methylation of Cx26may play a general role in the development of AHL, at least partially, by affecting the CpG sites of the promoter region of Cx26gene.