外周血NR3C1和NR3C2基因DNA甲基化、基因表达及交互作用与精神分裂症体力攻击行为的相关性研究

The prevalence of violence behavior in patients with schizophrenia is much higher than that in common population. It's not only a great threat to the safety of the patient，their family and the public but also a main cause of growing financial cost of the disease. For these reasons, violence in schizophrenia constitutes a major public health concern as well as social concern. The pathogenic mechanisms of aggressive behavior in schizophrenia patients have not been fully elucidated. In our previous study, we found that polymorphisms in genes related to the hypothalamic-pituitary-adrenal(HPA) axis were associated with different types of aggressive behavior in a differential way，which indicated that epigenetic regulation might play an role in the development of aggression. One of the major challenges in current psychiatric epigenetic studies is the tissue specificity of epigenetic changes since access to brain samples is limited. Several recent studies reported that blood-based biomarkers can predict aspects of brain signaling.The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play important roles in the hypothalamic-pituitary-adrenal axis and stress regulation. Based on these findings, a case-control study is designed to evaluate if DNA methylation of genes (NR3C1 and NR3C2) coding for glucocorticoid receptors(GR and MR respectively) can affect aggressive behavior using peripheral blood. To minimize the effect of unknown confounders such as antipsychotics, only patients with first-episode untreated schizophrenia can be recruited into this study, and only physical aggressive behavior is studied. In this study, DNA methylation status, mRNA expression level and protein expression level of the NR3C1 and NR3C2 will be detected. Statistical analysis will include association between DNA methylation status and physical aggression, association between gene expression levels and physical aggression, effects of DNA methylation on gene expression as well as effects of gene-gene interaction on physical aggression. Findings of this study will contribute to explain etiology of physical aggression in schizophrenia patients, and may provide theoretical basis for prediction,intervention,and treatment of physical aggression in schizophrenia patients as well as provide targets for future development of new drug. Therefore, it is of great scientific and socio-economic importance.

精神分裂症攻击行为高于普通人群，严重危害患者自身、家庭及社会公共安全,造成沉重的经济负担，是当前备受关注的公共卫生问题和社会问题，其形成机制目前尚不清楚。本项目组前期研究发现，HPA轴基因多态与攻击行为相关，但相同的遗传易感基因对不同攻击表型影响不同，提示表观遗传可能参与了调控，结合文献报道外周血中存在反映中枢神经系统改变的标记物，本项目拟采用病例-对照研究策略，以首发未治精神分裂症患者为研究对象，针对体力攻击表型，检测外周血中HPA轴激活终末产物——糖皮质激素的受体基因（NR3C1和NR3C2）DNA甲基化状态、mRNA表达和蛋白质表达，探讨NR3C1和NR3C2基因表观遗传变化、基因表达及交互作用对体力攻击行为的影响，为阐明精神分裂症体力攻击行为的形成机制提供理论依据，为精神分裂症攻击行为风险预测、干预治疗以及临床药物设计等提供新思路，具有重要的科学意义和社会经济意义。

精神分裂症患者比普通人及其他精神疾病更容易发生暴力攻击行为，并具有突发、攻击对象不确定、手段残暴的特点，对患者自身、家庭、医护及社会安全造成巨大威胁，但其发生机制迄今尚不清楚。本研究以以首发未治精神分裂症患者（伴/不伴体力攻击行为）和健康个体为研究对象，采用高通量测序技术对外周血中NR3C1 和 NR3C2基因启动子区域的DNA的甲基化水平进行检测分析, RT-qPCR和ELISA方法分别检测基因mRNA和蛋白表达水平，双荧光素酶报告基因实验检测DNA甲基化对转录活性的影响，5’-杂氮胞苷处理研究去甲基化对细胞内源性基因mRNA表达的影响，并在SH-SY5Y细胞中研究基因敲低对细胞生长、增值影响，并以RNAseq分析敲低基因后的差异表达基因，GO和KEGG分析初步探讨NR3C1和NR3C2基因可能发挥的生物学功能。结果发现：①NR3C1和NR3C2两个基因的启动子区域的DNA甲基化均与精神分裂症攻击行为密切相关，多个片段和位点在SCZ攻击组与非攻击组之间甲基化水平具有显著性差异，并存在性别特异性。②两个基因在SCZ攻击组的mRNA和蛋白表达水平均高于SCZ非攻击组，在SCZ非攻击组中的表达水平低于健康对照组。③NR3C1和NR3C2基因的甲基化会抑制基因的转录活性，去甲基化处理后细胞内源性基因mRNA表达上调。④ NR3C1和NR3C2基因敲低后SH-SY5Y细胞神经突明显延长，并且能够促进增殖。转录组测序结果显示基因敲低后分别引起388和296个差异基因表达，其中有72个差异表达的基因有交集。GO和KEGG分析发现差异表达基因主要参与细胞连接、生物调节、对应激的反应、发育等过程，富集在趋化因子介导的信号通路、MAPK、发育过程与环境信息处理相关的信号转导、细胞增殖，生长和死亡及神经退行性疾病等过程。上述研究发现为阐明精神分裂症体力攻击行为的发生机制提供了理论依据，筛选出的的显著性片段及位点可作为精神分裂症体力攻击行为潜在生物标志物，并为其干预及药物设计提供了新思路。