miR-184对小鼠神经干细胞增殖的影响及其机制  

Effect of miR-184 on proliferation of neural stem cells and its underlying mechanism

作　　者：黄佛宝 张昊驹 刘钰罡 罗丹[1] 徐如祥 戴宜武 

机构地区：[1]南方医科大学第二临床学院,广州510150 [2]安徽医科大学研究生学院,合肥230032 [3]陆军总医院附属八一脑科医院,北京100700

出　　处：《中华神经医学杂志》2017年第3期262-268,共7页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81271391、30840082）

摘　　要：目的观察miR-184对小鼠神经干细胞（NSCs）增殖的影响及其机制。方法构建包装pHBLV-U6-GFP-miR-184基因过表达和pHBLV—U6-GFP-sh-miR-184基因干扰慢病毒载体和各自对照无义序列慢病毒载体，分离培养孕14d的CD1胎鼠大脑侧脑室下区的NSCs并鉴定。实验细胞分为过表达对照组、miR-184过表达组、干扰对照组和miR-184干扰组，各组细胞在感染对应的慢病毒后，用嘌呤霉素进行抗性筛选。筛选后，继续培养细胞至3代，实时定量PCR进行miR-184过表达验证。通过targetScan、iRTarBase、miRanda等软件预测 miR—184的靶基因并进行内源性验证，采用5-溴脱氧尿嘧啶核苷（Brdu）细胞渗入实验观察各实验组的增殖情况，通过Western blotting实验和实时定量PCR观察各组Notch信号通路相关蛋白Hes1和Hes5及其mRNA的表达情况。结果免疫荧光染色显示，90％的分离培养细胞呈巢蛋A（nestin）和Y性别决定转录因子2（Sox2）双阳性，miR-184过表达组的miR—184表达量是过表达对照组的（67．63±7．53）倍，差异具有统计学意义（P〈0．05）。Brdu细胞渗入实验结果显示与各自对照组比，miR-184过表达组的细胞增值率是过表达对照组的（1．47±0．05）倍，而miR—184干扰组的细胞增值率是干扰对照组的（0．84±0．03）倍．差异均具有统计学意义（P〈0．05）。iRTarBase和miRanda软件预测，Numblike蛋白（Numbl蛋白）作为miR-184的潜在靶基因，miR—184可以下调Numbl蛋白的表达，miR-184过表达组和miR-184干扰组的Numbl相对表达量分别是各自对照组的（0．73±0．07）倍、（1．30±0．05）倍，差异均具有统计学意义（P〈0．05），但miR—184并不能改变Numbl mRNA的表达水平。miR-184可抑制Numb1蛋白的表达．使Notch信号通路的下游相关蛋白Hes1和Hes5及其mRNA表达升高，差异均具有统计学意义（P〈0．05）。结论miR-184通过在蛋白翻译水平抑制Numb1�Objective To investigate the effect ofmiR-184 on proliferation of neural stem cells （NSCs） and its mechanisms in mice. Methods The pHBLV-U6-GFP-miR-184 over-expression plasmid and pHBLV-U6-GFP-miR-184 inhibitor plasmid were used to construct recombinant lentivirus. And the NSCs derived FRom subventricular zone of E14d CD1 mouse were confirmed by immunofluorescence assay. There were four groups that contain a miR-184 overexpression group, a miR-184 inhibitor group and two control groups. The NSCs which infected with lentiviral vectors were selected for puromycin resistance for 5-7 days, and then surviving cells were cultivated to three generations. The expression level ofmiR-184 was detected by real time-quantitative PCR （RT-qPCR）. And the target genes of miR-184 were predicted through TargetScan, IRTarBase and MiRanda, and were confirmed by Western blotting and RT-qPCR. The cells in the four groups were cultured under proliferating conditions incorporated bromodeoxyuridine （BrdU） in cell proliferation analyses. The protein expressions of Hes 1 and Hes5, the target proteins of Notch signaling pathways, and their mRNA expressions were detected by Western blotting and RT-qPCR. Results There were 90% of cells in each group expressing both Nestin and Sox2. The miR-184 level in the miR-184 overexpression group was 67.63±7.53 times of that of the control group, with significant difference （P〈0.05）. The percent of BrdU＋/DAP1＋ of the miR-184 overexpression group was 1.47±0.05 times of that in the control group, with significant difference （P〈0.05）; and the percent of BrdU＋/DAPI＋ of the miR-184 inhibitor group was 0.84±0.03 times of that in the inhibitor control group, with significant difference （P〈0.05）. Numbl was a target gene of miR-184 indicated by IRTarBase and MiRanda. The miR-184 could inhibit Numbl protein expression; the Numbl protein expression level in the miR-184 overexpression group was 0.73±0.07 times of that in the control group, and the Numbl protein expression lev

关 键 词：miR-184 神经干细胞 Numbl蛋白 NOTCH信号通路 Hesl蛋白 Hes5 蛋白 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]