| 史晓东,郑娇琳,焉春华,田佳楠,李 慧,马 煦,王晓坤,聂雪丹,杨春晓.MiR-381下调Hes1表达调控神经干细胞增殖和向神经元的分化[J].,2017,17(20):3821-3826 |
| 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 |
| 投稿时间：2017-01-04 修订日期：2017-01-28 |
| DOI：10.13241/j.cnki.pmb.2017.20.005 |
| 中文关键词: 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) |
| 英文关键词: Neural stem cells Neuron miRNA-381 Hes1 |
| 基金项目:黑龙江省卫生计生委科研课题（2016-071） |
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| 中文摘要: |
| 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bFGF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。 |
| 英文摘要: |
| ABSTRACT 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 under 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, β-tubulin-III and Hes1 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 Hes1 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 Hes1 vector was significantly reduced by miR-381 over expression, however, the 3'UTR of mutant Hes1 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 mRNA and protein expression of β-tubulin III, were down-regulated by miR-381 and Hes1 co-transfection compared with that of miR-381 single transfection group (p < 0.01, p < 0.001). Conclusion: MiR-381 promotes the proliferation of NSCs and the differentiation into neurons via down-regulating Hes1 expression. |
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