耳蜗毛细胞miR-30b对Dynamin基因靶向调控的研究

Expression and Regulatory Effect of miR-30 bon Dynamin in Cochlear Hair Cells

目的观察正常成年小鼠耳蜗毛细胞中miR-30b的表达,研究其对靶基因(Dynamin1,DNM1)的调控是否会影响内毛细胞中突触囊泡内吞关键蛋白Dynamin的表达。方法取正常成年C57小鼠耳蜗基底膜行原位杂交,观察耳蜗毛细胞中miR-30b的表达分布;构建荧光素酶报告基因载体,转染293T细胞,通过荧光素酶活性变化明确DNM1是否为miR-30b的靶基因;通过耳蜗圆窗显微注射过表达miR-30b的腺相关病毒(adenoassociated virus,AAV),14d后通过real time-PCR检测miR-30b和DNM1的mRNA表达,免疫印迹实验(Western blot)检测Dynamin蛋白表达。结果 miR-30b在小鼠耳蜗内、外毛细胞胞质和胞核均有表达;miR-30b可抑制DNM1载体的荧光表达(P<0.05),而突变型DNM1载体的荧光表达无抑制作用;转染AAV-miR-30b后miR-30b的相对表达量增高,DNM1和Dynamin蛋白表达降低。结论 miR-30b在小鼠耳蜗内、外毛细胞均有表达;miR-30可以靶向下调DNM1基因从而抑制Dynamin蛋白表达。

Objective To determine the expression of miR-30 bin hair cells of mice,and its regulatory effect on the target gene DNM1 and expression of Dynamin,the key protein of synaptic endocytosis in inner hair cells.Methods The basilar membrane of cochlear in adult C57 mice was obtained.The expression of miR-30 bin the hair cells was detected by in situ hybridization.Luciferase vector was constructed and transfected into 293 Tcells with miR-30 b.Changes in luciferase activity were measured to verify whether DNM1 was the target gene of miR-30 b.Adeno-associated virus carrying miR-30 bwere micro-injected into cochlear via the round window membrane.mRNA expressions of DNM1 and miR-30 b were detected by RT-PCR 14 days later.The expression of Dynamin was detected by Western blot.Results miR-30 bexpressed in the inner and outer hair cells scattered in the region of the nucleus and cytoplasm.miR-30 breduced luciferase activity from the reporter vector containing DNM1（P〈0.05）,but not in its mutants.Increased expressions of miR-30 band decreased mRNA expressions of DNM1 and Dynamin were observed following transfection of AAV-miR-30 b.Conclusion miR-30 bexpresses in inner and outer hair cells,which is consistent with the morphological orientation of dynamin.miR-30 binhibits the expression of Dynamin by targeting DNM1 gene.