摘要
目的研究ApoE基因型是如何影响脑源性神经营养因子(brain derived neurotrophic factor,BDNF)的表达,以更好的理解AD的发病机制。方法在SH-SY5Y细胞系和大鼠原代神经元中加入Apo E3和Apo E4重组蛋白,用real-time PCR、Western blot和ELISA检测BDNF的表达水平;用luciferase assay、real-time PCR和Western blot证实BDNF是mir-30a-5p的靶基因;用Taq Man技术检测mir-30a-5p的水平。结果与对照组相比,Apo E3增加了BDNF的mRNA、蛋白水平和培养基中的BDNF浓度,而ApoE4减少了BDNF的mRNA、蛋白水平和培养基中的BDNF浓度; hsa-mir-30a-5p抑制BDNF的3’UTR的luciferase活性,mRNA和蛋白水平。ApoE3降低mir-30a-5p的表达,Apo E4增加mir-30a-5p的表达,而过表达mir-30a-5p inhibitor可以逆转ApoE4对BDNF的抑制效果。结论BDNF是mir-30a-5p的靶基因,ApoE是通过影响mir-30a-5p的水平来调控BDNF的表达。
Objective To investigate how ApoE genotype affects brain derived neurotrophic factor( BDNF) expression in the pathogenesis of AD. Methods ApoE3 and ApoE4 recombinant proteins were added to SH-SY5 Y and rat primary neurons,the expression of BDNF was detected by real-time PCR,Western blot and ELISA. The luciferase assay,real-time PCR and Western blot confirm that BDNF is the target gene of mir-30 a-5 p. Results Compared to the control group,ApoE3 increased both BDNF mRNA and its protein levels as well as BDNF concentration in the culture medium while ApoE4 reduced BDNF at mRNA and protein levels as well as BDNF concentrations in the culture medium. At the same time,hsa-mir-30 a-5 p inhibits luciferase activity,mRNA and protein levels of the 3'UTR of BDNF. ApoE3 increased BDNF expression by decreasing mir-30 a-5 p,while overexpression of mir-30 a-5 p mimic reversed the effect of ApoE3 on BDNF. ApoE4 decreased BDNF expression by increasing mir-30 a-5 p,while overexpression of mir-30 a-5 p inhibitor reversed the inhibitory effect of ApoE4 on BDNF. Conclusion BDNF is a target gene for mir-30 a-5 p,and ApoE regulates the expression of BDNF by affecting the level of mir-30 a-5 p.
引文
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