长链非编码RNA分子BC088414在发育期缺氧缺血脑损伤中的作用及机制

Neonatal hypoxic-ischemic encephalopathy (HIE) is a serious neurological disease in children during developing period. It is very important to explore the injury and reparation mechanism of HIE. In the preliminary work, we have found that 322 long non-coding RNAs (lncRNAs) were differently expressed between hypoxic-ischemic (HI) and normal brain tissues screened by RNA chip. Among them BC088414 is the one with the maximum differential expression. Silencing of BC088414 in PC12 cell, a cell line derived from a pheochromocytoma of the rat adrenal medulla, decreased PC12 cell apoptosis, suggesting BC088414 participates in the regulation of cell apoptosis (these results have been published). When comparing the sequences of BC088414 and microRNAs through bioinformatics analysis, we found that the sequence of BC088414 contains the binding site of miR-30d-5p. Then we predicted the targeted genes of miR-30d-5p using Targetscan and microRNA.org database, it showed that some apoptosis related genes such as caspase 6 were targeted by miR-30d-5p. Therefore, we proposed that BC088414 might regulate HI induced neuronal apoptosis as the competing endogenous RNA (ceRNA) of microRNAs. We hypothesize that when BC088414 binds to microRNAs such as miR-30d-5p, the inhibition effect of miR-30d-5p on gene expression will be attenuated, consequently the expression of miR-30d-5p targeted genes such as caspase 6 will be up-regulated, followed by enhanced neuronal apoptosis and brain damage induced by HI. To prove this hypothesis, in the present study, we will first construct HI induced neuronal damage model in vitro and rat brain damage model in vivo. Then using these models, we’ll try to clarify the regulation effect of BC088414 on the microRNAs and targeted genes through ceRNA pathway, examine the effect of lncRNA regulating strategy on attenuating HI induced brain damage in neonatal rat, and explore the best therapeutic time window, finally provide new ideas for HIE therapy and prevention.

新生儿缺氧缺血性脑病（HIE）是发育期严重疾病，探索其损伤机制意义重大。我们前期在新生鼠缺氧缺血脑组织中筛选得到差异表达的长链非编码RNA(lncRNA)BC088414，发现其能调控细胞凋亡（已发表）。继而采用生物信息学分析,发现BC088414序列包含microRNA（miR-30d-5p等）结合位点，提示BC088414可能为特异microRNA的竞争性内源RNA（ceRNA）。进一步分析发现，miR-30d-5p靶基因包含Casp6等凋亡相关基因, 强烈提示BC088414可作为ceRNA削减microRNA对其靶基因的抑制，上调凋亡相关基因表达，促进神经元凋亡。为证实此设想，本课题将建立体内外缺氧缺血神经损伤模型，探讨BC088414对特异microRNA及其靶基因的调节机制，并对lncRNA调控策略用于减轻发育期脑损伤的效果进行评价，确定最佳治疗时间窗，为HIE治疗提供新思路。

新生儿缺氧缺血性脑病是引起新生儿死亡和脑瘫的主要原因，其神经细胞损伤机制尚不完全清楚。长链非编码RNA（lncRNAs）和microRNAs（miRNAs）是调控基因表达的重要分子。miRNA抑制基因表达，而lncRNAs可以作为miRNAs的ceRNA，解除miRNA对靶基因的抑制作用，从而促进基因表达。我们前期研究发现，miR-30d-5p是促进发育期缺氧缺血脑损伤细胞凋亡的重要因素。资助项目计划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，探讨lncRNA 分子BC088414作为ceRNA对miR-30d-5p的靶基因的调控作用，并探索应用lncRNA调控策略减轻新生大鼠缺缺血脑损伤的效果。. 项目执行过程中，我们发现氧糖剥夺（OGD）处理后，大鼠体外神经元中BC088414和Casp6的mRNA均表达上调。然而，BC088414主要在细胞核中表达，提示它对Casp6的表达调控可能通过蛋白质作用途径而非ceRNA机制。随着大鼠lncRNA数据库的不断更新，BC088414被认定为编码RNA。为了从lncRNA角度探讨miR-30d-5p在缺氧缺血脑损伤中的作用及机制，我们重新利用生物信息学分析预测与miR-30d-5p相互作用的lncRNA分子，经StarBase在线数据库分析，发现PVT1中具有miR-30d-5p的结合位点，因而我们着重于研究PVT1分子。由于小鼠的lncRNA数据库比较成熟，我们改用了小鼠建立缺氧缺血脑损伤模型，并探讨PVT1作为miR-30d-5p的ceRNA在新生小鼠缺氧缺血脑损伤中的作用及具体机制。. 通过深入研究，我们在体外培养神经元氧糖剥夺模型及新生小鼠缺氧缺血脑损伤动物模型中，证实PVT1通过miR-30d-5p的ceRNA促进钙调蛋白依赖性激酶IIδ（CamKIIδ）的表达，参与调控缺氧缺血脑损伤诱导的神经元死亡。利用寡核苷酸分子ASO敲低PVT1导致缺氧缺血引起的神经元死亡增多，小鼠脑损伤加重，表明PVT1在缺氧缺血脑损伤中是一个神经保护分子，为探索新生儿缺氧缺血脑病治疗措施提供了新思路。