靶向骨形态发生蛋白4的miR-340-5p对大鼠神经干细胞分化的影响

Effect of miR-340-5p targeting at BMP4 on differentiation of neural stem cells in rats

目的探讨miR-340-5p对靶点骨形态发生蛋白4（bonemorphogeneticprotein4，BMP4）的调控作用及对大鼠神经干细胞（NSCs）分化的影响。方法传至第3代的SD大鼠NSCs随机为空白对照组（Mock）、无义寡核苷酸组（Anti—Con）和miR-340-5p反义寡核苷酸组（Anti-miR-340-5p），采用荧光定量PCR（qRT-PCR）检测miR-340—5p的表达，应用免疫细胞化学和免疫荧光方法分别观察NF200和MAP2表达，运用Westernblot法检测BMP4蛋白表达情况。结果Anti-miR．340-5p组miR-340-5p的相对表达量为（0．14±0．01），较Mock组（1．01±0．17）和Anti—Con组（1．07±0．13）显著降低（P〈0．01）；Anti—miR-340—5p组细胞NF200呈强阳性表达；miR-340—5p反义寡核苷酸转染组MAP2阳性细胞百分比显著高于Anti—Con组（P〈0．05）；与Mock组（0．53±0．04）和Anti·Con组（0．63±0．09）相比，An-ti—miR-340-5p组BMP4蛋白相对表达量显著升高（0．84±0．09）（P〈0．05）。结论miR-340-5p可能通过负性调控BMP4表达，发挥潜在的调控离体培养的大鼠NSCs向神经元分化功能。

Objective To explore regulation effect of miR-340-Sp on regulating bone morphogenetic protein 4 （BMP4） expression and the differentiation of rat＇ s NSCs. Methods NSCs of rats were divided randomly into three groups： normal group （Mock） , group with nonsense oligonueleotide （Anti-Con） and group with antisense oligonucleotide of miR-340-Sp （Anti-miR-340-Sp）.The qRT-PCR was used to detect the relative expression of miR-340-Sp. The expression of NF200 and MAP-2 was detected by immunocyto- chemical staining and immunofluorescenee, respectively. Western blot was used to detect the expression of BMP4 protein. Results The relative levels of miR-340-Sp expression were significantly decreased in Anti- miR-340-Sp group （0.14±0.01） compared with that of Mock group（ 1.01±0.17 ） and Anti-Con group （ 1.07± 0.13） （P〈0.01）. Immunoeytochemical staining indicated that NF200 was positive in cells of Anti-miR-340- 5p group. The proportion of MAP2 positive cells was increased in Anti-miR-340-Sp group compared with oth- er groups （P〈 0. 05 ） . Western blot showed the increased expression of BMP4 protein in Anti-miR-340-Sp group （0.84±0.09） compared with Mock group（0.53±0.04） and Anti-Con group （0.63±0.09） （P〈0.05）. Conclusion The miR-340-Sp may exert a potential function in regulating differentiation of NSCs into neurons through a negative regulation of BMP4.