摘要目的探讨miR-340-5p对靶点骨形态发生蛋白4(bone morphogenetic protein 4,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Western blot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,Anti-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.

abstractsObjectiveTo explore regulation effect of miR-340-5p on regulating bone morphogenetic protein 4(BMP4) expression and the differentiation of rat's NSCs.MethodsNSCs of rats were divided randomly into three groups: normal group (Mock),group with nonsense oligonucleotide (Anti-Con) and group with antisense oligonucleotide of miR-340-5p (Anti-miR-340-5p).The qRT-PCR was used to detect the relative expression of miR-340-5p.The expression of NF200 and MAP-2 was detected by immunocytochemical staining and immunofluorescence,respectively.Western blot was used to detect the expression of BMP4 protein.ResultsThe relative levels of miR-340-5p expression were significantly decreased in Anti-miR-340-5p 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).Immunocytochemical 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-340-5p group compared with other groups (P<0.05).Western blot showed the increased expression of BMP4 protein in Anti-miR-340-5p group (0.84±0.09) compared with Mock group(0.53±0.04) and Anti-Con group (0.63±0.09) (P<0.05).ConclusionThe miR-340-5p may exert a potential function in regulating differentiation of NSCs into neurons through a negative regulation of BMP4.