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Mir-27a promotes apoptosis of cochlear sensory epithelium in Cx26 knockout mice
Yunfeng Wang, Chen Lin, Yingzi He
1Department of Otolaryngology, Affiliated Eye and ENT Hospital of Fudan University, Building 9 Room 611, 83 Fenyang Road, Xuhui District, Shanghai 200031, China, huawli@sina.cn.
Abstract:
To investigate the underlying molecular mechanism for connexin 26 (Cx26) knockout-induced apoptosis, we performed TUNEL assays to detect apoptosis in the cochlear sensory epithelium in Cx26 knockout mice. We also compared the miRNA expression profiles of Cx26 knockout and wild-type mice using microarray technology and bioinformatic analyses. Real-time PCR, luciferase reporter gene assays, and scala media microinjections were performed to identify the effect of a specific miRNA and its targets. The results showed that apoptosis increased in the cochlear sensory epithelium of Cx26 knockout mice. The abnormal expression of mir-27a and sgk1 in Cx26 knockout mice was verified with real-time PCR. Luciferase reporter gene assays showed that overexpression of mir-27a significantly decreased sgk1 reporter gene activity; an inhibitor of mir-27a blocked the effect. Mir-27a lentivirus also inhibited sgk1 expression in cultured cochlear tissue. Mir-27a shRNA treatment inhibited Cx26 knockout-induced apoptosis in the cochlear sensory epithelium of mice and increased the expression of sgk1 mRNA. Thus, mir-27a was identified as an apoptotic molecule that participates in Cx26 knockout-induced apoptosis in the cochlear sensory epithelium of mice by downregulating sgk1 expression.
Insights
Connexin 26 (Cx26) knockout increases apoptosis in mouse cochlear sensory epithelium. Mir-27a, a microRNA, drives this apoptosis by downregulating sgk1 expression, revealing a key molecular mechanism.
Area of Science:
- Otolaryngology
- Molecular Biology
- Genetics
Background:
- Connexin 26 (Cx26) plays a crucial role in hearing.
- Cx26 deficiency is linked to hearing loss and cellular dysfunction.
- The molecular pathways driving Cx26 knockout-induced apoptosis remain unclear.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying apoptosis in the cochlear sensory epithelium of Cx26 knockout mice.
- To identify specific microRNAs (miRNAs) and their targets involved in this process.
Main Methods:
- TUNEL assays for apoptosis detection in Cx26 knockout mice.
- Microarray analysis of miRNA expression profiles.
- Real-time PCR, luciferase reporter assays, and microinjections to validate miRNA-target interactions.
- In vivo and in vitro experiments using lentivirus and shRNA to manipulate mir-27a and sgk1.
Main Results:
- Apoptosis was significantly increased in the cochlear sensory epithelium of Cx26 knockout mice.
- Mir-27a expression was upregulated, while sgk1 expression was downregulated in Cx26 knockout mice.
- Mir-27a directly targets and downregulates sgk1 expression.
- Mir-27a inhibition or sgk1 restoration reduced Cx26 knockout-induced apoptosis.
Conclusions:
- Mir-27a acts as an apoptotic mediator in Cx26 knockout-induced cochlear injury.
- The mir-27a/sgk1 axis is a critical molecular pathway involved in Cx26 deficiency-related apoptosis.
- Targeting mir-27a may offer a therapeutic strategy for hearing loss associated with Cx26 dysfunction.
