Piwil2/piRNA介导CREB2基因启动子甲基化在大鼠短暂全脑缺血耐受中的机制

Non-coding RNA (ncRNA), a functional RNA molecule that regulates gene expression at the transcriptional and post-transcriptional level, is emerging as an essential player in the pathogenesis of many diseases. As the largest class of small ncRNA molecules expressed in animal cells, Piwi-interacting RNAs (piRNAs) form RNA-protein complexes through interaction with PIWI proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements through de-novo DNA methylation. Our previous studies demonstrated that the expression of Piwil2 protein can be upregulated in the hippocampal CA1 subregion after transient global cerebral ischemia (tGCI), while hypoxic postconditioning (HPC) with 8% O2 decreased Piwil2 expression and ameliorated neuronal damage in CA1 after tGCI. However, the role of the Piwil2/piRNA complexes in ischemic tolerance against tGCI and the underlying molecular mechanisms still need to be further elucidated. Based on our previous findings, we hypothesized that HPC can offer neuroprotection against tGCI by downregulating Piwil2/piRNA complexes, thereby increasing the expression of CREB2 by inhibiting DNA methylation of CREB2 promoter, and eventually led to the enhancement of neuroplasticity associated with memory function and the attenuation of apoptotic neuronal death. In this project, we aimed to verify this hypothesis at molecular, cellular, organic and systemic level adopting state-of-the-art approaches. Taken together, this innovative project is anticipated to contribute to elucidate gene regulatory mechanisms mediated by Piwil2/piRNA complexes in ischemic tolerance against tGCI in adult rats, which will further lay the foundation for using Piwil2/piRNA as a potential biomarker or therapeutic target in tGCI.

非编码RNA在基因表达调控中具有重要作用。作为一类数目最大、新发现的小非编码RNA，piRNA（Piwi-interacting RNA）与PIWI蛋白结合，通过DNA甲基化等表观遗传修饰，参与调控mRNA的稳定性和蛋白质的合成等。我们前期研究发现，予8%O2的低氧后处理通过下调海马CA1区的Piwil2，使Piwil2/piRNA复合物减少，从而发挥神经保护作用。然而，该复合物对脑缺血及缺血耐受的影响，迄今仍不完全清楚。为此，我们提出假设，低氧后处理通过调控短暂全脑缺血后CA1区Piwil2/piRNA复合物，使CREB2基因启动子甲基化降低，CREB2表达增加，增强与记忆相关的神经突触可塑性，减轻神经元凋亡。本课题通过分子、细胞、组织及动物水平等加以证实，将揭示Piwil2/piRNA复合物在脑缺血耐受中的作用机制，并为其作为脑缺血诊断的生物标记物和药物治疗靶点奠定理论基础。

近年的研究表明，表观遗传修饰与脑缺血后的病理生理机制密切相关。非编码RNA在基因表达调控中具有重要作用。作为一类数目最大、新发现的小非编码RNA，piRNA（Piwi-interacting RNA）与PIWI蛋白结合，通过DNA甲基化等表观遗传修饰，参与调控mRNA的稳定性和蛋白质的合成等。然而，Piwil2/piRN复合物如何在脑缺血耐受中发挥作用，迄今仍不清楚。本项目在成年大鼠短暂全脑缺血低氧后处理模型的基础上，主要采用荧光定量PCR、免疫印迹、RNA免疫共沉淀、甲基化特异性PCR、基因测序分析、高尔基染色和行为学评估等技术，探讨Piwil2/piRNA复合物在脑缺血耐受中的作用机制。结果显示：（1）HPC抑制大鼠tGCI后CA1区Piwil2的表达；经侧脑室注射Piwil2反义寡核苷酸（antisense oligonucleotide，ASODN），可使tGCI后CA1区Piwil2的表达下调，存活的神经元显著增多；而经双侧CA1区注射过表达Piwil2的Lenti-Piwil2，可抵消低氧后处理对大鼠tGCI后CA1区神经元的保护作用。（2）高通量测序结果显示，tGCI组与HPC组CA1区存在差异性表达的piRNAs，功能分析结果显示，差异表达的piRNAs与轴突形成及突触功能相关；与tGCI组比较，在HPC组明显表达下调的3种piRNAs的共同靶基因为CREB2。（3）HPC通过抑制tGCI后CA1区Piwil2的表达，下调DNMT3A，降低CREB2基因启动子甲基化，增加CREB2 mRNA及蛋白表达，抑制缺血后CA1区的细胞凋亡，增强与记忆相关的神经突触可塑性，改善学习和记忆能力，发挥神经保护作用。通过本项创新研究，首次揭示了Piwil2/piRNA复合物在脑缺血耐受中的作用机制，并为其作为脑缺血诊断的生物标记物和药物治疗靶点奠定理论基础。