RESULTS: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student\'s t test was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05.
CONCLUSIONS: If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.
结果:在SH-SY5Y细胞中进行的荧光素酶实验表明,Br活性被REST选择性地降低,而Ht活性被DNMT1、MeCP2和REST降低。值得注意的是,Ht-RE1的定点突变阻止了Ncx1的REST依赖性下调。此外,在短暂大脑中动脉闭塞的8周龄雄性野生型小鼠(C57BL/6)的颞顶皮层中,DNMT1、MeCP2和REST与Ht启动子的结合增加,随之而来的DNA启动子超甲基化。侧脑室注射siREST可防止DNMT1/MeCP2结合Ht和Ncx1下调,从而减少中风引起的损伤。始终如一,在接受氧和葡萄糖剥夺加复氧的皮质神经元中,Ncx1敲低抵消了去甲基化剂5-氮杂胞苷诱导的神经元保护作用。对于两个实验组之间的比较,使用学生的t检验,而对于两个以上的实验组,使用了一元方差分析,其次是Tukey或NewmanKeuls.P<0.05时具有统计学意义。
结论:如果这项研究的结果在人类中得到证实,可以断言DNMT1/MeCP2/REST复合物破坏可能是减少大脑中HtDNA甲基化的新药理学策略,改善中风损伤。