siRNA沉默Cdkl5对原代皮质神经元Mecp2及其下游靶基因表达的影响

The effect of Cdkl5 shRNA on the expression of Mecp2 and its related downstream target genes in primary cortical neurons

目的探讨CDKL5在Rett综合征中与Me CP2相互作用的机制。方法体外培养孕18 d SD胎鼠皮质神经元,通过小RNA干扰技术(siRNA)敲低Cdkl5,分为干扰组(Cdkl5 shRNA)和对照组(control shRNA)。用Real time-PCR及Western blot法评估Cdkl5在mRNA和蛋白水平被敲低的效率;Cdkl5表达降低后Mecp2和Mecp2相关的下游靶基因表达变化。对Cdkl5 shRNA转染后神经元用CCK8法检测吸光度值评估神经元活力;用real time-PCR和Western blot法检测凋亡基因caspase3和凋亡活性蛋白cleaved-caspase3评估神经元凋亡状态。结果干扰组与对照组比较,Cdkl5在mRNA水平下降73%,在蛋白水平下降75%(P<0.05);Mecp2在mRNA和蛋白水平升高约2倍(P<0.05);BdnfⅪ在mRNA水平明显下降,Psd95在mRNA水平明显上升(P<0.05)。结论建立稳定的Cdkl5基因敲低的原代皮质神经元模型,并初步提示CDKL5可能通过对Me CP2的调控诱导了下游靶基因的变化。

Objective To investigate the relationship between CDKL5 and MeCP2 in the mechanism of Rett syndrome.Methods The primary cortical neurons obtained from embryonic day 18 Sprague Dawley rat were transfected by effective small interfering.RNAs to silence Cdkl5,dividing into Cdkl5 shRNA group and control shRNA group.RT-PCR and Western blot were used to evaluate the efficiency of silenced gene and detect its influence on both Mecp2 and the related downstream target genes.The cell viability was assessed by the value of optical density through CCK8.The state of cell apoptosis was investigated by the level of caspase3 and cleaved-caspase3 through real timePCR and Western blot.Results Compared with control shRNA group,transfection with effective Cdkl5 shRNA in neurons decreased Cdkl5 mRNA by 73％ and Cdkl5 protein by 75％ （P ＜ 0.05）.Cdkl5 inhibition was related to more than 2-fold induction of Mecp2 both in mRNA and protein level （P ＜ 0.05）.At the same time,the level of Bdnf Ⅺ transcript decreased while Psd95 increased in transfected neurons （P ＜ 0.05）.Conclusions A stable model of Cdkl5 silenced neurons has been established.The results suggest that CDKL5 may fulfill its duty on downstream target genes through regulating the function of MeCP2.