microRNA-183调节FOXO1对听觉毛细胞再生作用机制研究
The Mechanism of MicroRNA-183 Regulating FOXO1 on Auditory Hair Cell Regeneration
练东玲 1涂钰莹 1涂玉梅 1龙孝斌1
作者信息
- 1. 南方医科大学珠江医院耳鼻喉头颈外科/南方医科大学第二临床医学院(广州 510280)
- 折叠
摘要
目的 探讨microRNA-183(miR-183)对斑马鱼内耳生长发育的影响,及其在顺铂损伤内耳神经丘毛细胞及耳囊、耳石后的再生和生长发育中的作用.方法 利用显微注射技术向斑马鱼受精卵内分别注入miRNA模拟物(agomir)或反义吗啉寡核苷酸(MOs),初步构建miR-183过表达或低表达模型,然后将受精后第72 h斑马鱼浸泡在50μmol/L顺铂溶液中24 h完成最终建模.利用miRNA微阵列分析、RT-qPCR等技术检测miR-183和FOXO1基因表达的改变;通过FM1-43FX荧光染色了解内耳神经丘毛细胞损伤及再生情况;使用正置显微镜观察耳囊、耳石生长发育变化.结果 ①顺铂作用后斑马鱼内耳毛细胞受损且耳囊、耳石生长发育受阻;移除顺铂后内耳毛细胞可再生,耳囊、耳石生长发育也逐渐恢复.②miR-183在移除顺铂后被激活,是斑马鱼幼体miRNA中最有表达差异的成员之一(P＜0.001),且表达量持续上调至峰值后逐渐恢复至正常水平.③与对照组相比,移除顺铂后过表达miR-183组可促进内耳毛细胞数量和耳囊耳石面积的恢复(P＜0.01);低表达miR-183组则使其恢复受到抑制(P＜0.05).④移除顺铂作用后FOXO1被激活,表达量上调(P＜0.01);当过表达miR-183时FOXO1受到抑制,表达下调(P＜0.01);低表达miR-183时FOXO1则表达量上升(P＜0.01).结论 miR-183与移除顺铂作用后内耳毛细胞的再生、耳囊耳石面积生长发育的恢复呈正向调控关系,其机制可能与miR-183对FOXO1的负向调控有关.
Abstract
Objective To study the effect of microRNA-183(miR-183)on the growth and development of ze-brafish inner ear,and its mechanism in the regeneration and growth of inner ear hair cells,otocyst and otolith in-jured by cisplatin.Methods Synthesized miRNAs(agomir)and antisense morpholino oligonucleotides(MOs)were injected into zebrafish embryos by microinjection to construct the model of these miRNAs'levels.The 72-hours post fertilization zebrafish were immersed in 50 μmol/L cisplatin for 24 hours.MiRNA microarray analysis,RT-qPCR and other techniques were used to detect the changes of miR-183 and FOXO1 gene expression.FM1-43FX fluores-cence staining was used to understand the injury and regeneration of inner ear nerve thalamic hair cells.The micro-scope was used to observe the growth and development of otocyst and otoliths.Results ① After the treatment of cisplatin,the inner ear hair cells of zebrafish were damaged and the growth and development of otocyst and otoliths were hindered.After the removal of cisplatin,the inner ear hair cells could be regenerated,and the growth and de-velopment of otocyst and otoliths also recovered gradually.② miR-183 was activated after the removal of cisplatin,which was one of the most differentially expressed members in zebrafish larva miRNA(P＜0.001),and the expres-sion level increased continuously to the peak and then gradually returned to the normal level.③ Compared with the control group,after the removal of cisplatin,the overexpression of miR-183 could promote the recovery of the num-ber of hair cells and the area of otocyst and otoliths,while the recovery was inhibited in the group with low expres-sion of miR-183.④ After the removal of cisplatin,FOXO1 was activated and the expression was up-regulated(P＜0.01).When miR-183 was overexpressed,FOXO1 was inhibited and expression was down-regulated(P＜0.01).Conversely,when miR-183 was low,the expression of FOXO1 increased(P＜0.01).Conclusion The increase of miR-183 expression occurs simultaneously with the regeneration of inner ear hair cells and the recovery of growth and development of the otolith area after cisplatin removal.The overexpression or low expression of miR-183 can promote or inhibit the regeneration of inner ear hair cells and the recovery of growth and development of the otolith area,indicating that there is a positive regulatory relationship between them.The mechanism may be related to the negative regulation of miR-183 on FOXO1.
关键词
斑马鱼/顺铂/毛细胞/MicroRNA-183/FOXO1Key words
Zebrafish/Cisplatin/Hair cells/MicroRNA-183/FOXO1引用本文复制引用
出版年
2024
NETL
NSTL
万方数据