Long non-coding RNAs in alcohol induced neural cell death

酒精滥用会对大脑和行为产生破坏性影响，对公共卫生构成沉重负担。 然而，酒精诱导的神经细胞损伤的分子基础尚未完全了解。最近 功能基因组学的进展表明，长链非编码RNA（lncRNA）可能在酒精中发挥关键作用， 诱导神经细胞死亡。我们实验室最近的工作支持这一假设。因此 酒精诱导神经细胞死亡相关lncRNA的综合筛选体系具有重要意义 识别潜在治疗靶点的益处。 基因编辑工具，如锌指核酸酶（ZFN）和转录激活样元件 核酸酶（TALEN）在基因功能研究和基因治疗中具有巨大的应用潜力 通过敲除或敲入技术。一种基于细菌染色体的新型基因编辑工具 规则间隔短回文重复序列（CRISPR）/CRISPR相关系统[Cas]）已被 成功地用于从细菌和酵母到哺乳动物的各种生物体。相对于ZFN和TALEN， CRISPR/Cas是有利的，因为它只需要改变指导RNA（gRNA）的序列，并且它 也可以直接输送到胚胎中以产生基因修饰的生物体。此外，复用 CRISPR/Cas的能力使得同时靶向多个基因成为可能。 在这项研究中，我们将使用我们最近开发的双指导CRISPR/Cas方法来产生lncRNA。 在SH-SY 5 Y细胞模型中对gRNA基因敲除文库进行测序。然后我们会对lncRNAs进行全基因组筛查 参与酒精诱导的细胞死亡途径。我们的初步数据表明，酒精增加了 SH-SY 5 Y细胞中核旁斑lncRNA的表达。我们以前的工作已经证明， 酒精以剂量依赖性方式激活氧化应激-凋亡细胞死亡途径， 神经元细胞活力和增加细胞氧化应激。我们期望高通量的应用 酒精诱导的神经元死亡系统的筛选平台将允许准确识别所有其他 特异性lncRNA以及它们在介导酒精诱导的神经变性中的潜在作用。所生成的 本研究中的lncRNA gRNA文库也可用于表征由其他药物诱导的细胞毒性。 神经毒素或其他物质滥用。因此，这项研究有可能减少酒精对健康的影响。 滥用，也有利于其他物质滥用相关的公共健康负担。

Alcohol abuse can have devastating impacts on the brain and behavior and is a big burden on public health. However, the molecular underpinnings of alcohol induced neural cell injury are not fully understood. Recent advances in functional genomics suggest that long non-coding RNAs (lncRNAs) may play critical roles in alcohol- induced neural cell death. The recent work from our laboratory support this hypothesis. Therefore, a comprehensive screening system of lncRNAs related to alcohol-induced neural cell death is of significant benefits for identifying potential therapeutic targets. Genetic editing tools such as Zinc Finger Nuclease (ZFN) and Transcription Activation-Like Element Nuclease (TALEN) have great potential for the functional study of genes or the application of gene therapy through knockout or knockin techniques. A new type of genetic editing tool based on bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR‐Associated System [Cas]) has been successfully used in various organisms from bacteria and yeast to mammals. Relative to ZFN and TALEN, CRISPR/Cas is advantageous because it only requires changing the sequence of the guide RNA (gRNA) and it can also be directly delivered into embryos to generate gene-modified organisms. Furthermore, the multiplexing capability of CRISPR/Cas makes it possible to target multiple genes simultaneously. In this study we will use our recently developed dual guide CRISPR/Cas approach to generate an lncRNA knockout (gRNA) library in SH-SY5Y cell model. We will then perform a genome-wide screening for lncRNAs involved in alcohol-induced cell death pathways. Our preliminary data indicated that alcohol increased the expression of a nuclear paraspeckle lncRNA in SH-SY5Y cells. Our previous work have demonstrated that alcohol activates the oxidative stress-apoptotic cell death pathway in a dose dependent manner to reduce neuronal cell viability and increase cellular oxidative stress. We expect that the application of the high throughput screening platform in alcohol-induced neuronal death system will allow for the accurate identification of all other specific lncRNAs as well as their potential roles in mediating alcohol-induced neurodegeneration. The generated lncRNA gRNA library in this study can also be available for the characterization of cell toxicity induced by other neurotoxins or other substance abuse. Thus this research has a potential to reduce the health impacts of alcohol abuse and also has benefits for other substances abuse related public heath burdens.