伏隔核区lncRNA uc.84-调节可卡因成瘾及其分子机制研究

The molecular mechanism of drug addiction is one of the neuroscience research focuses. We first used a custom-designed microarray to examine the expression profiles of long non-coding RNAs (lncRNAs) in Nucleus Accumbens (NAc) of cocaine conditioned mice. We found uc.84-, a extremely conserved lncRNA in mammals, was significantly upregulated in NAc of cocaine conditioned mice. According to analysis of the genomic context and cis-/trans- of uc.84-, we found that it is located at the downstream 5.5kb of Nurr1. Nurr1 is a key transcription factor in dopaminergic neuron, which regulates dopamine transporter, tyrosine hydroxylase and other addiction-related genes. In this study, lentivirus uc.84- shRNA vector inject into NAc, RT-PCR, western blot, conditioned place preference and locomotor activity will be evaluated the effect of uc.84- on cocaine induced addiction and neural plasticity. siRNA transfection, dual luciferase reporter gene will be used to evaluate the enhancer effects of uc.84-. Overall, this study will be expected to clarify uc.84- regulates cocaine induced addiction and its molecular mechanism, which provides new strategy for the prevention and treatment of cocaine addiction.

药物成瘾的分子生物学机制是神经科学研究热点之一。我们首次通过高通量芯片对可卡因条件性位置偏好小鼠伏隔核进行了长链非编码RNA (lncRNA) 筛选。发现uc.84-，一个哺乳动物中高度保守的lncRNA，表达显著上调。进一步对uc.84-的基因组位置和Cis-/Trans-分析发现其位于转录因子Nurr1下游5.5kb处，并可能作为增强子促进Nurr1的转录。而Nurr1是多巴胺能神经元中的关键转录因子，调节多巴胺转运蛋白、酪氨酸羟化酶等多个成瘾相关基因表达。本课题拟通过uc.84- shRNA慢病毒伏隔核定位注射，RT-PCR、Western blot、动物行为学研究uc.84-对可卡因诱导成瘾行为和神经可塑性的影响；并通过siRNA转染、双荧光素酶报告法等技术研究uc.84-的增强子效应。通过本项研究，有望阐明uc.84-调节可卡因成瘾及其分子机制，为可卡因成瘾的防治提供新策略。

成瘾药物的滥用和依赖引起了全球性严重的医疗和社会问题。近年来，ncRNA在药物成瘾和神经突触可塑性研究中取得了很大进展，如miR-212放大CREB信号通路参与可卡因成瘾研究等，但药物成瘾中lncRNA分子生物学机制研究方面，仍属空白。本课题主要研究内容与结果如下：（1）建立了可卡因诱导CPP和自发活动模型，采用荧光定量PCR、Western blot等技术，发现可卡因给药组伏隔核uc.84-和Nurr1表达水平均显著升高，但在纹状体、海马和前额皮质等三个脑区的表达没有显著变化，表明uc.84-和Nurr1表达水平升高具有脑区特异性，仅在伏隔核区。（2）为研究伏隔核区uc.84-上调表达在可卡因成瘾中的作用，通过生物信息学预测和体外验证，构建了uc.84- shRNA的慢病毒。脑定位内注射于小鼠伏隔核，观察对可卡因诱导的CPP和自发活动的影响。神经行为学分析发现，伏隔核定位注射uc.84- shRNA可以显著抑制可卡因的CPP奖赏效应和行为敏化效应。另外发现伏隔核定位注射uc.84- shRNA能够降低可卡因诱导的DAT和TH表达的升高和树突棘密度增加，表明uc.84-参与可卡因成瘾与调控DAT和TH的表达有关。（3）采用荧光素酶报告基因系统研究发现，uc.84-能够作为增强子参与Nurr1表达的调控，参与可卡因成瘾的过程。本课题阐明了uc.84-参与可卡因成瘾形成的分子生物学机制，从lncRNA调控的角度诠释可卡因成瘾的形成原因，对探索药物成瘾新的靶标具有重要意义，这将对未来可卡因成瘾的防治提供新思路。