miR-204-5p 通过调控 SIRT1 促进 C57BL/6 耳聋小鼠内耳细胞内质网应激诱导的细胞凋亡
MiR-204-5p regulates SIRT1 to promote the endoplasmic reticulum stress-induced apoptosis of inner ear cells in C57BL/6 mice with hearing loss.
机构信息
Chongqing Medical University, Chongqing, China.
Department of Otolaryngology, Chongqing General Hospital, Chongqing, China.
出版信息
PLoS One. 2024 Nov 12;19(11):e0309892. doi: 10.1371/journal.pone.0309892. eCollection 2024.
PURPOSE
This study investigated the effect of miR-204-5p-mediated silencing of SIRT1 on the development of deafness in C57BL/6 mice and the roles of miR-204-5p and SIRT1 in deafness.
METHODS
Auditory brainstem response recordings, H&E staining, and immunohistochemistry were used to observe changes in hearing function and cochlear tissue morphology in 2-month-old and 15-month-old C57BL/6 mice. A senescence model was induced using H2O2 in inner ear cells (HEI-OC1). Changes in HEI-OC1 cell proliferation were detected using the CCK-8 assay, whereas flow cytometry was used to detect changes in apoptosis. MiR-204-5p expression was measured via RT‒qPCR. The SIRT1 agonist RSV and a miR-204-5p inhibitor were used to study changes in ER stress (ERS), proliferation, and apoptosis in HEI-OC1 cells. Western blotting was performed to detect changes in ATF4, CHOP, SIRT1, PERK, p-PERK, Bax, and Bcl-2 protein levels. A dual-luciferase reporter gene assay was carried out to assess the ability of miR-204-5p to target SIRT1.
RESULTS
Relative miR-204-5p expression levels in the cochleae of aged C57BL/6 mice increased, whereas SIRT1 expression levels decreased, and miR-204-5p and SIRT1 expression levels were negatively correlated. ERS and increased 8-OHDG levels were observed in aged C57BL/6 mice. In a model of inner ear cell aging, H2O2 treatment induced increases in miR-204-5p expression and ERS-mediated apoptosis. MiR-204-5p was found to target SIRT1 and inhibit its expression. SIRT1 activation and a miR-204-5p inhibitor promoted HEI-OC1 cell proliferation and reduced apoptosis. The miR-204-5p inhibitor regulated expression of the ERS proteins PERK, ATF4, and CHOP to upregulate Bcl-2 and downregulate Bax.
CONCLUSION
This study identified the roles of miR-204-5p and SIRT1 in deafness in C57BL/6 mice and investigated the loss of cochlear outer hair cells and the involvement of apoptosis and ERS in deafness.
目的
本研究旨在探讨 miR-204-5p 介导的 SIRT1 沉默对 C57BL/6 小鼠耳聋发生的影响及其在耳聋中的作用。
方法
采用听觉脑干反应记录、H&E 染色和免疫组织化学方法观察 2 月龄和 15 月龄 C57BL/6 小鼠听力功能和耳蜗组织形态的变化。采用 H2O2 诱导内耳细胞（HEI-OC1）衰老模型。通过 CCK-8 法检测 HEI-OC1 细胞增殖变化，流式细胞术检测细胞凋亡变化。采用 RT‒qPCR 检测 miR-204-5p 的表达。使用 SIRT1 激动剂 RSV 和 miR-204-5p 抑制剂研究 HEI-OC1 细胞中 ER 应激（ERS）、增殖和凋亡的变化。采用 Western blot 检测 ATF4、CHOP、SIRT1、PERK、p-PERK、Bax 和 Bcl-2 蛋白水平的变化。采用双荧光素酶报告基因检测 miR-204-5p 对 SIRT1 的靶向作用。
结果
老年 C57BL/6 小鼠耳蜗中相对 miR-204-5p 表达水平升高，SIRT1 表达水平降低，且 miR-204-5p 与 SIRT1 表达水平呈负相关。老年 C57BL/6 小鼠中观察到 ERS 和 8-OHDG 水平升高。在内耳细胞衰老模型中，H2O2 处理诱导 miR-204-5p 表达增加和 ERS 介导的细胞凋亡。发现 miR-204-5p 靶向 SIRT1 并抑制其表达。SIRT1 激活和 miR-204-5p 抑制剂促进 HEI-OC1 细胞增殖并减少凋亡。miR-204-5p 抑制剂调节 ERS 蛋白 PERK、ATF4 和 CHOP 的表达，上调 Bcl-2，下调 Bax。
结论
本研究确定了 miR-204-5p 和 SIRT1 在 C57BL/6 小鼠耳聋中的作用，并研究了耳蜗外毛细胞的丧失以及凋亡和 ERS 在耳聋中的参与。
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