转基因心脏过表达microRNA-1通过分泌机制诱发小鼠认知功能障碍及其分子机制研究

Senile dementia is a close relationship with cardiovascular diseases, but the mechanism is unkown. In the present application, based on clinical issues, the effect of miR-1 with overexpression in the heart on learning and memory will be evaluated by bioinformatics, transgenic mice, locked nucleotide, miR-masking and other molecular biology techniques as well as water maze systerm. To elucidate the mechanism of cardiac desease induced cognitive impairment. Study contains: 1. To assess the behavioral characteristics and the expression of target proteins from Tg mice with miR-1 overexpression in heart, and detect miR-1 level in all of the heart, blood, cerebrospinal fluid, hippocampus and cortex; 2.To dectect the miR-1 level in hippocampal neurons after co-cultured with myocardial cells; 3. To evaluate the expression of target proteins in primary hippocampal neurons after transfection with miR-1, AMO-1,NC or ODNs of target genes, and determine the precise regulational relationship; 4. To assess the behavioral characteristics and the expression of target proteins from Tg mice after LNA-antimiR-1 and lentiviral vector administration. This study is the first time to report that transgenic overexpression of microRNA-1 in heart could cause cognitive dysfunction in mice by its secretion. This application will unveil a new biological significance of microRNAs.

老年期痴呆与多种心血管系统疾病密切相关，但机制不清。本研究立足临床现象联合应用网络数据库、转基因动物、锁核苷酸、miR-masking及相关分子生物学技术配合水迷宫检测系统探讨转基因心脏过表达miR-1（Tg）对小鼠学习记忆的影响。阐明心脏疾病诱发认知功能障碍的microRNA机制。研究内容：1.评价Tg小鼠行为学特征和目的蛋白的表达，检测miR-1在其血液、脑脊液、心脏、海马以及皮层的变化;2.共培养技术检测心肌细胞过表达miR-1后海马神经元miR-1的表达；3.检测细胞水平转染miR-1、AMO-1、NC及目的蛋白ODNs后目的蛋白的变化及其直接的调控关系;4.评价LNA-antimiR-1全身干预以及慢病毒载体局部海马注射对小鼠行为学特征及目的蛋白的影响。本研究首次提出心脏miR-1过表达通过分泌机制引起认知功能障碍，揭示microRNAs新的生物学意义。

老年期痴呆与多种心血管系统疾病密切相关，但机制不清。本研究立足临床现象联合应用网络数据库、转基因动物、锁核苷酸、miR-masking及相关分子生物学技术配合水迷宫检测系统探讨转基因心脏表达miR-1（Tg）对小鼠学习记忆的影响。阐明心脏疾病诱发认知功能障碍的microRNA机制。研究内容：1.评价Tg小鼠行为学特征和目的蛋白的表达.检测miR-1在血液、脑脊液、心脏、海马以及皮层的变化;2. 细胞共培养技术检测原代培养心肌细胞过表达miR-1后海马神经元miR-1的表达；3.检测细胞水平转染miR-1、AMO-1、NC及目的蛋白ODNs后目的蛋白的变化及其直接的调控关系;4.评价LNA-antimiR-1全身干预以及慢病毒载体局部海马注射对小鼠行为学特征及目的蛋白的影响。 研究结果表明：1.小鼠心肌梗死引起miR-1表达升高。升高的miR-1可以通过血液循环进入海马，抑制微管聚合蛋白TPPP的表达诱发海马神经元微管的溶解。2. 心脏特异性过表达miR-1的Tg小鼠海马组织miR-1表达水平升高。心脏特异过表达miR-1 Tg小鼠的认知功能下降。MiR-1通过抑制脑源性神经营养因子（BDNF）蛋白的表达进而抑制cAMP反应元件结合蛋白（CREB）的磷酸化诱导小鼠认知功能下降3. 脏特异性过表达miR-1的Tg小鼠海马神经元递质释放障碍。MiR-1可以直接抑制递质释放相关的SNAP-25蛋白的表达。心脏特异性过表达miR-1的Tg小鼠海马高表达的miR-1可以抑制海马神经元的递质释放。本研究首次提出心脏miR-1过表达引起认知功能障碍，揭示microRNAs新的生物学意义。