中文摘要：

目的探讨在耳蜗毛细胞氧化损伤中表达异常的miR-181a对原癌基因c-fos的生物学调控作用。方法采用叔丁基过氧化氢（t-BHP）染毒，设50、100、200μmol／L 3个浓度组和未染毒对照组对HEI-CO1细胞染毒12h，提取细胞总RNA及细胞总蛋白，采用荧光定量PCR（qPCR）检测miR-181a／-181d表达；选择表达异常的miR-181a为研究对象，检索生物信息学网站，对c-fos 3′端非翻译区域（3′-UTR）上miR-181a可能的作用靶序列进行预测分析；用脂质体转染入miR-181a的mimics，qPCR检测转染效率；qPCR观察c-fos mRNA水平表达量的改变；免疫印迹（Western blotting）法观察毛细胞中c-fos蛋白表达量的改变；构建野生型的融合c-fos-3′UTR的重组pGL3质粒（pGL3-c-fos-3′UTR-WT），利用双荧光素酶报告基因检测系统检测在高表达miR-181a后荧光素酶活力的改变情况。结果qPCR结果显示，miR-181a在100、200μmol／L染毒浓度组表达下调，低表达倍数分别为0.744和0.766，差异均有统计学意义（P〈0．01）；而miR-181d在50μmol／L浓度组表达升高，高表达倍数为1．29，差异亦有统计学意义（P〈0．01），在100、200μmol／L 2个染毒浓度组中的表达变化的差异无统计学意义（P〉0．05）；通过预测显示，在人类和小鼠中，c-fos均受miR-181a的调控，在种子区域完全互补，靶序列在物种之间也十分保守；脂质体转染入miR-181a mimics 24h后，qPCR结果显示，在miR-181a mimics转染组中miR-181a升高892．979倍；与对照组相比，在miR-181a mimics转染组的耳蜗毛细胞中c-fos mRNA的表达增高，差异有统计学意义（P〈0．05）；Western blotting观察结果显示，与对照组相比，c-fos蛋白在miR-181a mimics转染组中表达升高，差异有统计学意义（P〈0．01）；成功构建pGL3-c-fos-3′UTR-WT的重组质粒，双荧光素报告基因检测系统结果显示，在高表达miR-181a时，野生型pGL3-c-fos-3′U

英文摘要：

Objective To investigate the regulatory effect of miR-181a with abnormal expression on the expression of c-fos in cochlear hair cells undergoing oxidative damage. Methods House Ear Institute- Organ of Corti 1 （HEI-CO1） cells were assigned to 50, 100, and 200 μmol/L tert-butyl hydroperoxide （t-BHP） exposure groups and control group. The HEI-CO1 cells in the exposure groups were exposed to 50, 100, or 200 μmol/L t-BHP for 12 h. Then, total RNA and total protein were extracted from the HEI-CO1 cells, and the expression of miR-181a/-181d was measured by qPCR. The miR-181a with abnormal expression was selected as the subject of study. The putative miR-181 a target sequence in the 3′ untranslated region （3′-UTR） of c-fos was predicted by searching on a bioinformatics website. The HEI-CO1 cells were transfected with miR-181a mimics by lipofection, and the transfection efficiency was measured by qPCR. The mRNA and protein expression of c-fos was measured by qPCR and Western blot. The pGL3-c-fos-3′UTR-WT plasmid was constructed, and the luciferase activity of the plasmid in the case of high miR-181a expression was measured using the Dual-Luciferase Reporter Assay System. Results Compared with those in the control group, the expression of miR-181a in 100 and 200 μmol/L t-BHP exposure groups was significantly decreased, with expression ratios of 0.744 and 0.766 （P〈0.01 ）, while the expression of miR-181 d in 50 μmol/L t-BHP exposure group was significantly increased, with an expression ratio of 1.29 （P〈0.01）. There was no significant difference in miR-181a expression between the 100 and 200 μmol/L t-BHP exposure groups （P〉0.05）. The predication results revealed that c-fos was regulated by miR-181a in humans and mice, with complete complementarity to the seed region of miR-181a, and there was high degree of target sequence conservation across species. The expression of miR-181a in the HEI-OC1 ceils transfected with miR-181a mimics was elevated 892.979 times at 24 hours after transfect