转录因子Bhlhb2在ADHD中的调节网络研究

Based on our previous results, we are going to investigate the regulation roles in ADHD and extract total RNA from the right prefrontal cortex tissue of ADHD rats to analyse the expression profiles of both mRNA and miRNAs, and screen our interested genes and miRNAs which are differentially expressed. Through the bioinformatics analysis, their common transcription factors wil have been predicted in cross regulation analysis. Then the realtime PCR, Western blot and luciferase reporter assay were performed to verificate the results of chip and the prediction, and the transcription factor Bhlhb2 will on the center of the regulation network. Furthermore, the project further tests the regulation effect of transcription factors (SP1, Cux1, etc) on the expression of Bhlhb2 and miRNAs (138, 138 *) and the regulation effect of miRNAs (138, 138 *) on the expression of Bhlhb2. To validate the regulation effect of the Bhlhb2 on Bdnf, tranfection of Bhlhb2 expression and interference plasmids will be performed in PC12 cells. Detecting the expression of Bhlhb2 and Bdnf in ADHD rats brain tissue is to analyze the relationship between their expression levels in ADHD. All the regulation pathways of Bhlhb2 regulation by Cux1 and miRNA-138, etc in the proposal have not been reported, and the significance of these regulations remains unknown yet. Our research about the Bhlhb2 will provide important experiment basis to understand the roles of Bhlhb2 in the pathogenesis of ADHD.

根据项目前期的实验结果，抽提ADHD大鼠右侧前额组织RNA，进行mRNA和miRNAs表达谱的分析，筛查具有差异表达的基因和miRNAs，筛选分析，并对二者进行交集分析，预测它们共同的转录因子；通过realtime PCR、蛋白质免疫印记电泳和荧光素酶报告系统检测，验证上述芯片以及预测的结果，筛查得到Bhlhb2转录因子；项目还进一步检测转录因子（SP1,Cux1等）对Bhlhb2以及miRNAs（138,138*等）的表达影响，检测miRNAs（138,138*等）对Bhlhb2的表达影响。在PC12细胞转染Bhlhb2表达和干扰质粒，验证Bhlhb2对Bdnf的影响，并在ADHD大鼠脑组织测定Bhlhb2、Bdnf，分析其表达水平与ADHD的关系。项目所研究的调控关系均未有报告，研究结果将为理解ADHD的发病机制提供重要的实验依据。

背景. 注意缺陷多动障碍（ADHD）是全球最常见的儿童行为障碍，其病因和发病机制仍不清楚。 通过全面的研究 miRNA /mRNA 的相互作用网络，发现在多动症动物模型自发性高血压大鼠（SHR）的前额叶皮质（PFC）中一个调控Bhlhb2和脑源性神经营养因子（BDNF）的复杂的TF-miRNA网络。其在多动症发病中起到非常重要的作用。.研究内容、结果. 比较SHR与对照组WKY大鼠的主要脑区中NR3C1、NF1、Bhlhb2和BDNF表达差异。利用mRNA、蛋白质和microRNA差异表达谱确定了糖皮质激素受体（NR3C1）和神经纤维瘤蛋白1（NF1）两个重要的转录因子。与对照组相比，SHR大鼠PFC中NR3C1表达上调，NF1的表达下调。NR3C1抑制miR-138-1，34C，296，和494基因的启动子活性并抑制它们的转录，而NF1促进miR-138-2基因的表达。NR3C1的异位表达增强Bhlhb2基因的启动子活性。过表达Bhlhb2抑制BDNF的表达，而抑制Bhlhb2可提高BDNF表达。体内实验发现，SHR大鼠PFC中Bhlhb2的敲低可明显改善其多动症症状。SHR大鼠血液中皮质酮和患有ADHD的男孩血液中皮质醇浓度较正常对照组低，与大脑中的NR3C1的高表达一致。.科学意义. 研究发现了多动症大鼠的PFC中一个复杂的TF-miRNA网络，NR3C1-Bhlhb2轴的失调影响多动症的发生发展，是参与ADHD发病的重要机制。