靶向HGTD-P的microRNA鉴定及其抑制新生大鼠缺氧缺血神经元凋亡研究

Neonatal hypoxic-ischemic encephalopathy(HIE)occurs in 1-6 of 1000 live newborn babies and is one of the major causes of neonatal death worldwide. Moreover, survivors of HIE may have various neurological impairments, including mild learning disabilities, mental retardation, cerebral palsy, motor deficits, and epilepsy. Therefore, it is very important to explore the pathogenic and repair mechanism of HIE. Our research recently published on "Stroke" showed that the up-regulation of human growth and transformation dependent protein(HGTD-P) is a key factor to promote neuronal apoptosis induced by hypoxia-ischemia in neonatal rat. Since HGTD-P peaks at 36 to 48 hours after hypoxia-ischemia, it may contribute to the protracted period of brain damage, and the late activation of HGTD-P suggests an extended therapeutic window for intervention of HIE. However, the mechanism how HGTD-P is regulated in nervous system remains unknown.In the preliminary study, we have predicted microRNAs targeted to HGTD-P through bio-informatics, we found that some of them overlapped with the microRNAs differently expressed between hypoxic-ischemic and normal brain tissues screened by microRNA chip,suggesting that the variation of some specific microRNAs is the crucial mechanism to up-regulate HGTD-P in hypoxic-ischemic neuron.In the present study, we'll try to select and identify microRNAs targeted to HGTD-P, and prove that these microRNAs are new molecules which up-regulate HGDT-P in hypoxic-ischemic neuron through molecular experiment，in vitro cell research and animal testing. Meanwhile, since microRNAs have higher stabilization and better tissue compatibility, we 'll explore the possibility and feasibility of using microRNAs to inhibit neuronal apoptosis induced by hypoxia-ischemia in neonatal rat.

新生儿缺氧缺血性脑病（HIE）是小儿严重的神经系统疾病，探索HIE病理及修复机制具有重要意义。我们近期发表在"Stroke"杂志的研究表明，人生长与转化依赖蛋白（HGTD-P）表达增加，是促进新生鼠缺氧缺血后神经元凋亡的重要因素。前期工作中，.我们经生物信息学预测了可能靶向调控HGTD-P基因的microRNA,发现其与microRNA表达谱芯片筛查到的缺氧缺血和对照脑组织差异表达的microRNA有重叠，推测某些特异性的microRNA表达变化可能是缺氧缺血神经元上调HGTD-P基因表达的重要机制。本项目拟通过分子、细胞和动物实验筛选鉴定与HGTD-P相互作用的microRNA分子，证明其为缺氧缺血神经元上调HGTD-P基因表达的新分子。同时，考虑到microRNA具有更高的稳定性和更好的组织相容性，我们将进一步探索应用microRNA分子抑制新生大鼠缺氧缺血神经元凋亡的可能性及可行性。

新生儿缺氧缺血往往导致严重的脑损伤。在新生脑中，缺氧缺血性脑损伤（HIBD）一般通过坏死或凋亡途径导致细胞死亡。目前，许多研究证实细胞凋亡途径在HIBD中更常见。HGTD-P是新近发现的促凋亡蛋白，是缺氧缺血细胞死亡的效应分子。研究发现，发育中的大鼠脑缺氧缺氧后，HGTD-P通过激活半胱氨酸蛋白酶-3和促进凋亡诱导因子核易位，发挥重要的促凋亡作用。为了抑制HGTD-P表达及其诱导的细胞凋亡，我们需要阐明缺氧缺血上调HGTD-P表达的机制。microRNA（miRNA）是近年来发现的调控基因表达的重要分子,一些miRNA已被证实受缺氧的诱导，并参与缺氧诱导的细胞或组织生物行为的改变。鉴于此，我们假设某些特定miRNA的变化可能是HIBD中HGTD-P表达变化的潜在调控机制。本研究中,我们通过生物信息学预测了靶向HGTD-P基因的14个microRNA分子。在体外培养神经元氧糖剥夺模型及新生大鼠脑缺氧缺血动物模型中，筛选鉴定出miR-139-5p通过靶向HGTD-P参与调控缺氧缺血诱导的神经元凋亡。进一步研究发现，缺氧缺血12h后应用miR-139-5p 激动剂可以减轻脑损伤，与现存的治疗方法相比较，提供了一个较长的治疗时间窗。