LincRNA在CYP46A1基因第二内含子T/C多态性对AD易感性影响中的作用及机制

A single nucleotide polymorphism (SNP) T/C (rs754203) in intron 2 of Cholesterol 24S-hydroxylase gene (CYP46A1) has been identified to be significantly associated with the susceptibility of Alzheimer’s disease (AD), but the mechanisms have remained unclear. We have known that decreased expression of CYP46A1 could increase the production and deposition of amyloid-beta (Aβ) peptides that result in the formation of amyloid plaques, the pathology of AD. CYP46A1 gene can transcript long intronic non-coding RNA (LincRNA) RP11-543C4.3-001 whose transcribed sequences locate in intron 2 of CYP46A1 and contain rs754203. Thus, we hypothesized that RP11-543C4.3-001 LincRNA could regulate gene expression of CYP46A1 and production of Aβ, and rs754203 would influence the regulatory function of RP11-543C4.3-001 LincRNA. We will observe expression of CYP46A1 and production of Aβ after increasing exogenous LincRNA and silencing endogenous LincRNA specifically by siRNA in human neuroblastoma cells, aiming to investigate the role of RP11-543C4.3-001 LincRNA in regulating expression of CYP46A1and production of Aβ. In addition, we will explore the regulatory mechanisms of RP11-543C4.3-001 LincRNA according to comparing the effects of two type of LincRNA on promotor and alternative pre-mRNA splicing, and studing different structure and target molecules in two type of LincRNA. Through these studies, we will ultimately clarify the role of RP11-543C4.3-001 LincRNA in the association between rs754203 polymorphism of CYP46A1 gene and the susceptibility of AD. Our study will be the first exploring the role of LincRNA in the pathogenesis of AD, and the results can provide theoretical evidences for revealing a new AD pathogenesis and a new target for gene therapy of AD.

胆固醇-24S羟化酶编码基因CYP46A1第二内含子T/C多态性（rs754203）与阿尔茨海默病（AD）易感性密切相关，其机制不详。CYP46A1表达水平降低可增加Aβ的产生，参与AD的病理形成。我们发现CYP46A1基因rs754203所在区域存在长链内含子非编码RNA（LincRNA）的转录序列，推测rs754203通过改变该LincRNA对CYP46A1基因表达的调控作用影响AD易感性。本项目以人神经母细胞瘤细胞为模型，通过上调及下调该LincRNA研究其对CYP46A1基因表达及Aβ产生的调控作用；通过比较两型LincRNA的结构和靶分子，及其对CYP46A1启动子活性和mRNA剪接的影响探讨作用机制，最终阐明LincRNA在rs754203对AD易感性影响中的作用及机制。本项目首次在AD发病机制中探讨LincRNA的作用，为揭示AD新的发病机制及基因治疗新靶点提供有效理论依据。

阿尔茨海默病（AD）是环境因素与遗传因素共同作用的结果。APOE基因是目前唯一公认的散发性AD易感基因，提示脑内胆固醇代谢可能与AD关系密切。脑内胆固醇堆积，会影响脂筏分布，增加β淀粉样蛋白（Aβ）的产生，参与AD的病理形成。脑内胆固醇需要通过胆固醇24S-羟化酶（CYP46A1）将其转化为24S-羟胆固醇（24-OHC），进而通过血脑屏障进入外周血循环代谢。故CYP46A1编码基因可能与APOE一样是AD的易感基因。既往我们的meta分析已证实了CYP46A1第二内含子rs754203 T/C多态性与AD易感性的关系，该位点所在部位正好位于长链内含子非编码RNA（LincRNA）转录序列RP11-543C4.3中，LincRNA具有对母本基因表达的调控作用。我们提出假说：RP11-543C4.3-001 LincRNA对CYP46A1基因表达具有调控作用；CYP46A1基因第二内含子T/C多态性改变了LincRNA的转录水平、结构或靶分子，进而影响LincRNA的调控功能；CYP46A1基因的AD危险基因型（CC或TT）所转录的LincRNA使CYP46A1基因表达水平降低，进而导致脑内胆固醇累积，增加Aβ的产生及沉积，最终参与AD的发生。.本项目围绕上述假说开展研究，取得如下结果：（1）证实了LincRNA对CYP46A1表达及功能具有抑制作用，对Aβ的产生具有促进作用；（2）证实了CYP46A1基因第二内含子T/C多态性可改变RP11-543C4.3-001 LincRNA对CYP46A1的调控功能，G型LincRNA对CYP46A1的抑制作用可能弱于A型LincRNA；（3）在中国人群中CYP46A1 C等位基因携带即增加AD风险，APOEε4可增强其对AD的风险性；（4）血浆24-OHC可作为AD早期诊断的生物标志物。.本研究首次对CYP46A1基因中的LincRNA（RP11-543C4.3-001）进行功能研究，并对CYP46A1第二内含子rs754203 T/C多态性影响AD发生的机制进行探讨。目前初步证实了LincRNA对母本基因CYP46A1表达具有抑制作用，rs754203 T/C多态性可能通过影响LincRNA对CYP46A1表达的抑制作用发挥功能。