c-Myc调控Id2在缺血缺氧性脑损伤中的机制及其干预研究

Further reveal the pathophysiological mechanism of ischemia/hypoxic-induced brain injury and improve the effect of its intervention therapy are important and difficult to neuroscience researchers. Through the researches granted by National Science Foundation, applicant previously discovered and confirmed that the expression of Id2 (inhibitor of DNA binding 2) was up-regulated significantly in neuronal cells after the exposure of hypoxic/ischemia, ptomoted the neuronal apoptosis; Biochemical experiments reveal more Id2 plays pivotal control of neurons in the Rb/E2F1 pathways. Further preliminary experiments found that c-Myc can combine with Id2 promoter regions, regulate the transcription of Id2; c-Myc’s nuclear localization also unusually increased in ischemia/hypoxic-induced brain injury; Application of c-Myc specific small molecule inhibitors (10058-F4) could reduce ischemia/hypoxia after brain infarct size, implicate Id2 is likely to be regulated by c-Myc in the ischemia/hypoxic-induced brain injury. Based on these research results above, the applicant intends to further research a new mechanism that c-Myc regulates Id2 in hypoxic/ischemia-induced brain injury and its intervention study the through the molecules, cells and animal research. Provides new scientific theory for ischemia/hypoxic-induced brain injury; Not only overcome the difficulty of Id2 small molecular inhibitors’ absence, c - Myc specific small molecule inhibitors also provides a new feasible method for clinical intervention treatment.

深入揭示缺血缺氧性脑损伤的病理生理机制、提高其干预治疗效果一直是神经科学领域的研究重点和难点。申请者在前期国家自然科学基金的研究发现并证实了Id2(inhibitor of DNA binding 2)蛋白在缺血缺氧性神经元中表达显著上升，促使了神经元的凋亡；生化实验更揭示Id2对神经元中Rb/E2F1通路的枢纽调控作用。进一步的预初实验研究发现c-Myc能够结合到Id2的启动子区域，调控Id2的转录；且在脑缺血缺氧损伤中，c-Myc核定位亦呈现异常升高；而c-Myc特异的小分子抑制剂（10058-F4）减小了缺血缺氧后脑梗塞面积，提示Id2在缺血缺氧性脑损伤中极有可能受到c-Myc的上游调控作用。拟进一步通过分子、细胞及动物的研究，深入探讨c-Myc在缺血缺氧性脑损伤中对Id2的作用机制及其干预研究，克服了Id2靶向抑制剂缺乏的困难，还为缺血缺氧性脑损伤提供新的科学理论和新的干预手段。

深入揭示缺血缺氧性脑损伤的病理生理机制、提高其干预治疗效果一直是神经科学领域的研究重点和难点。申请者在前期国家自然科学基金的研究发现并证实了Id2(inhibitor of DNA binding 2)蛋白在缺血缺氧性神经元中表达显著上升，促使了神经元的凋亡；生化实验更揭示Id2对神经元中Rb/E2F1通路的枢纽调控作用。本课题从细胞、动物和分子信号通路水平三方面深入探讨了c-myc对Id2在缺血缺氧诱导神经凋亡中的作用及其调控机制。.一方面，在c-myc对Id2调控方面：体内实验研究发现：在大鼠MCAO缺血/再灌注模型中，随着缺血缺氧时间的延长，脑梗死面积逐渐扩大；随后的分子实验证实与Id2类似，c-Myc核定位亦呈现异常升高；在CoCl2处理B35细胞模拟低氧培养的模型中，c-Myc mRNA及蛋白水平均随着低氧处理时间显著增高；荧光素酶报告基因实验提示c-Myc能够结合到Id2的启动子区域，调控Id2的转录；进而在体内实验，大鼠MCAO缺血/再灌注模型中，通过大鼠尾静脉试验性应用10058-F4（c-Myc特异的小分子抑制剂）能够减小了缺血缺氧后脑梗塞面积。.另一方面课题组在预实验探索中意外发现长链非编码RNA H19通过竞争性内源性RNA机制调控Id2。具体如下：1.抑制H19能够降低Id2的表达。在糖氧剥夺模型（oxygen glucose deprivation (OGD)）中，当H19被敲低，Id2的mRNA和蛋白表达均降低。流式细胞分析与免疫染色均揭示：敲低H19后，OGD神经元细胞的凋亡明显降低。2.miR-19a能够直接结合Id2。软件预测发现miR-19a可以结合Id2的3’-UTR，同时，过表达miR-19a能够抑制Id2 mRNA和蛋白的表达，抑制miR-19a时，Id2的mRNA和蛋白表达增加。双荧光素酶实验表明：miR -19a mimic能够降低Id2-wt的荧光素酶活性。3.H19通过竞争结合miR-19a，解除miR-19a对Id2的抑制。H19下调降低转染Luc-Id2-3’-UTR的HEK293T细胞荧光素酶活性。相反，miR-19a抑制剂的引入挽救了荧光素酶活性。此外，挽救实验也证实这一机制。.本研究重点探讨了在缺血缺氧诱导的神经细胞凋亡中Id2的上游调控机制，可为临床治疗脑缺血缺氧性疾病寻求新的治疗方法及潜在的治疗靶点。