Identification of miRNAs that regulate tinnitus development

To investigate which miRNAs regulate the development of tinnitus, we have performed microarray for miRNAs as a discovery platform. The changes in dorsal cochlear nucleus after noise exposure have been suggested to play an important role in the development of tinnitus. This study was performed using 12 week-old-male Sprague-Dawley rats. Based on our previous experiment about the development of tinnitus, we set a time point for harvesting dorsal cochlear nuclei: 3 weeks after the exposure of 6-8 kHz narrow-band noise (110 dB SPL for 2 h) at right side. Eight animals were divided into two experimental groups: tinnitus group showing evidence of tinnitus (n = 5); and non-tinnitus group showing no evidence of tinnitus (n = 3) in the gap pre-pulse inhibition of acoustic startle reflex (GPIAS) recordings. Three animals of each group were anesthetized deeply and euthanized at 3 weeks post noise exposure. Dorsal cochlear nucleus at right side was harvested. Microarray for miRNAs was performed using the Affymetrix miRNA 4.0 microarray. Considering the fold change of normalized signal intensities between tinnitus and non-tinnitus groups, miR-375-3p was selected as the candidate miRNA. This result was validated by quantitative reverse transcription-PCR.为探究调控耳鸣发生的微小核糖核酸（miRNAs），本研究以miRNA微阵列作为发现平台开展相关实验。已有研究证实，噪声暴露后耳蜗背核的分子变化在耳鸣发病进程中发挥关键作用。本研究选用12周龄雄性斯普拉格-道利大鼠（Sprague-Dawley rats）作为实验动物。基于前期关于耳鸣发生的实验基础，本研究设定噪声暴露后3周为耳蜗背核取材时间点：对大鼠右侧耳部给予6-8 kHz窄带噪声刺激，参数为110分贝声压级（dB SPL）、暴露时长2小时。通过声惊反射间隙前脉冲抑制（gap pre-pulse inhibition of acoustic startle reflex, GPIAS）检测记录，将8只大鼠分为两组：表现出耳鸣阳性特征的耳鸣组（n=5），以及未检测到耳鸣阳性特征的非耳鸣组（n=3）。每组各取3只大鼠于噪声暴露后3周深度麻醉并处死，采集其右侧耳蜗背核组织。本研究采用Affymetrix miRNA 4.0微阵列芯片平台完成miRNAs的表达谱检测。结合耳鸣组与非耳鸣组间标准化信号强度的倍数变化情况，筛选出miR-375-3p作为候选miRNA，并通过定量反转录聚合酶链反应对该筛选结果进行了验证。