MiR-381下调Hes1表达调控神经干细胞增殖和向神经元的分化  

MiR-381 Promotes the Proliferation and Differentiation of Neural Stem Cell via down-regulating Hes1 Expression

作　　者：史晓东[1] 郑娇琳 焉春华[2] 田佳楠[1] 李慧[1] 马煦[1] 王晓坤[1] 聂雪丹[1] 杨春晓[1] SHI Xiao-dong ZHENG Jiao-lin YAN Chun-hua TIAN Jia-nan LI Hui MA Xu WANG Xiao-kun NIE Xue-dan YANG Chun-xiao(Department of Neurology, the Second Affiliated Hospital of Harbin Medical Universify, Harbin, Heilongjiang, 150081, China Department of Respiratory, the Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150081, China)

机构地区：[1]哈尔滨医科大学附属第二医院神经科,黑龙江哈尔滨150081 [2]哈尔滨医科大学附属第二医院呼吸科,黑龙江哈尔滨150081

出　　处：《现代生物医学进展》2017年第20期3821-3826,3830,共7页Progress in Modern Biomedicine

基　　金：黑龙江省卫生计生委科研课题(2016-071)

摘　　要：目的:探讨miRNA-381在神经干细胞(neural stem cell,NSCs)的体外增殖、分化中的作用及相关机制。方法:采用光镜观察法、免疫组化法对原代获取及诱导分化后的NSCs予以鉴定;QPCR及Western blot法检测miRNA-381、nestin、β-tubulin-Ⅲ和Hes1的mRNA及蛋白表达;CCK-8法检测不同时间点NSCs的增殖情况;荧光素酶报告基因法验证miR-381对Hes1野生型及突变型3'非编码结合区的靶向作用。结果:原代培养细胞呈明显的神经球形态,且高表达nestin蛋白,经b FGF诱导后则高表达β-tubulin-Ⅲ;miR-381转染后,NSCs中miR-381、nestin、β-tubulin-Ⅲ的mRNA和蛋白水平均明显升高(p<0.001),免疫荧光法也进一步验证miR-381转染后的NSCs其β-tubulin-Ⅲ的荧光强度显著增强;此外,miR-381转染24 h、48 h及72 h后,NSCs的增殖能力均高于阴性对照组(p24h<0.01,p48h<0.001,p72h<0.001);荧光素酶法结果显示miR-381能显著降低野生型Hes1载体的3'非编码区的荧光素酶活性,而Hes1基因载体的突变型3'UTR的荧光素酶活性不受到miR-381的影响(p<0.001);另外,miR-381过表达能明显降低Hes1蛋白的表达;而二者共转染的NSCs增殖能力在转染后24 h、48 h及72 h,均明显低于单纯miR-381转染组(p<0.01或p<0.001);同时,共转染后β-tubulin Ⅲ的mRNA及蛋白水平均明显低于单纯miR-381转染组(p<0.001)。结论:miR-381通过下调Hes1表达促进NSCs的增殖和向神经元的分化。Objective：To explore the role of miRNA-381 in the proliferation and differentiation of neural stem cells （NSCs）,as well as the relative mechanisms.Methods：Observation unader light microscope and immunohistochemistry were used to identify NSCs.QPCR and Western blot were selected to detect both mRNA levels and protein levels of miRNA-381,nestin,β3-tubulin-Ⅲ and Hesl expression.The proliferation of NSCs was detected by CCK-8 assay,and Luciferase reporter gene assay was processed to measure effects of miR-381 on wild type/mutant 3＇UTR of Hesl vector.Results：Primary cultured neuronal cells showed typical morphology of neurosphere and highly expressed nestin protein,while cells presented high expression of β-tubulin-Ⅲ after bFGF induction.The mRNA and protein levels of miR-381,nestin,β-tubulin-Ⅲ in NSCs after miR-381 transfection were significantly higher than that of negative control group （p ＜ 0.001）,besides,the fluorescence intensity of β-tubulin-Ⅲ was also obviously enhanced.What＇s more,the proliferation of NSCs after 24 h,48 h and 72 h transfection by miR-381 was statistical higher than control group （p24h ＜ 0.01,p48h ＜0.001,p72h ＜0.001）;luciferase enzyme results showed that the luciferase activity of 3’UTR of wild-type Hesl vector was significantly reduced by miR-381 over expression,however,the YUTR of mutant Hesl vector was not influenced by miR-381 （p ＜ 0.001）;additionally,miR-381 over expression obviously reduced HES1 protein expression,and the proliferative ability of NSCs,as well as the rnRNA and protein expression of β-tubulin Ⅲ,were down-regulated by miR-381 and Hes 1 co-transfection compared with that of miR-381 single transfection group （p ＜ 0.01,p ＜ 0.001）.Conclusions：MiR-381 promotes the proliferation of NSCs and the differentiation into neurons via down-regulating Hes 1 expression.

关 键 词：神经干细胞 神经元 微小RNA-381 发状分裂相关增强子1(Hairy and ENHANCER of split 1 Hes1) 

分 类 号：R-33[医药卫生] Q42[生物学—神经生物学]