论文部分内容阅读
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 investigate the role of miRNA-381 in the proliferation and differentiation of neural stem cells (NSCs) in vitro and its related mechanisms. Methods: The primary NSCs obtained and differentiated were identified by light microscopy and immunohistochemistry. The mRNA and protein expressions of miRNA-381, nestin, β-tubulin-Ⅲ and Hes1 were detected by QPCR and Western blot. CCK-8 assay was used to detect the proliferation of NSCs at different time points. The luciferase reporter assay was used to validate the targeting of miR-381 to Hes1 wild-type and mutant 3 ’non-coding binding regions. Results: Primary cultured cells showed neurosphere morphology and high expression of nestin protein. After induced by b FGF, β-tubulin-Ⅲ was highly expressed. After transfection with miR-381, miR-381, nestin, β- The mRNA and protein levels of tubulin-Ⅲ were significantly increased (p <0.001), and the fluorescence intensity ofβ-tubulin-Ⅲ of miR-381 transfected NSCs was further verified by immunofluorescence. In addition, Proliferation of NSCs was higher than that of the negative control group (p24h <0.01, p48h <0.001, p72h <0.001) after 24 h, 48 h and 72 h of incubation. Luciferase assay showed that miR-381 significantly decreased the expression of wild type Hes1 Vector, whereas the luciferase activity of the mutant 3’UTR of the Hes1 gene vector was not affected by miR-381 (p <0.001). In addition, miR-381 overexpression was significantly (P <0.01 or p <0.001). However, the proliferation of NSCs co-transfection was significantly lower than that of miR-381 transfected cells at 24 h, 48 h and 72 h after transfection (p <0.01 or p <0.001) Meanwhile, the mRNA and protein levels of β-tubulin Ⅲ in co-transfection were significantly lower than those in miR-381 transfected group (p <0.001). Conclusion: miR-381 can promote NSCs proliferation and differentiation into neurons by down-regulating Hes1 expression.