Cannabinoid Receptor (CNR1) MicroRNAs and Addictive Disease

描述(申请人提供)：毒瘾是一种复杂的神经疾病，可能涉及遗传因素。例如，在老鼠身上的研究表明，编码大麻素受体的基因的消除会削弱它们对鸦片类药物和酒精的“成瘾反应”。尽管许多基因被怀疑与成瘾行为有关，但这些基因中明确促进药物成瘾易感性的独特的人类变异还没有确定。到目前为止，识别这类变异的大多数尝试都集中在可疑基因的蛋白质编码片段上。然而，改变这些候选基因表达的变异很可能也会使个体容易患上成瘾性疾病。最近，人们已经清楚地发现，许多人类基因受到一种新的表观遗传机制的调控，这种机制涉及到小RNA(MicroRNA)对信使核糖核酸中的靶成分进行特异性的退火。在大多数情况下，microRNA的退火会导致靶mRNA表达的显著下降。在我们的初步研究中，我们已经在人类大麻素受体mRNA的3‘UTR区发现了一个50核苷酸的元件，它很可能是microRNA的靶标。我们已经观察到，将该元件插入到荧光素酶报告基因中，显著降低了其表达。重要的是，靶分子的突变被预测为损害与microRNAs的结合，从而取消了靶分子的抑制作用。有趣的是，这一突变此前已被确认为非裔美国人群体中的一种罕见的多态。因此，我们的主要假设是大麻素受体受microRNAs调控，大麻素受体元件的变异可能会影响其表达，从而使个人对药物上瘾易感。在我们的第一个目标中，我们将确认和扩大我们的初步观察，并确定介导大麻素受体抑制的microRNA。这些研究将得到我们在设计和使用“安塔戈米尔”试剂方面的经验的帮助。反交配子是一种经过修饰的RNA，可以被引入细胞中，并能有效地下调特定microRNAs的表达。在我们的第二个目标中，我们将检查大麻素受体元件中的任何人类变异是否与成瘾易感性相关。为此，我们与Kranzler博士、Covault博士和Oncken博士建立了合作关系，他们可以从对照和受影响的患者群体中获取DNA。

DESCRIPTION (provided by applicant): Drug addiction is a complex neurological disorder likely to involve genetic components. For example, studies in mice have shown that elimination of the gene that encodes the receptor for cannabinoids blunts their "addictive response " to opiates and alcohol. Although many genes have been suspected to contribute to addictive behavior, unique human variants in these genes that unequivocally promote susceptibility to drug addiction have not been identified. So far, most attempts to identify such variants have focused on the protein coding segments of the suspect genes. However, it is quite possible that variants that alter the expression of such candidate genes may also render individuals susceptible to addictive disease. Recently, it has become clear that many human genes are regulated by a new epigenetic mechanism, which involves the specific annealing of a small RNA (microRNA) to a target element in the mRNA. In most cases, the annealing of the microRNA leads to a profound decrease in expression of the target mRNA. In our preliminary studies, we have identified a 50 nucleotide element in the 3'UTR of the human cannabinoid receptor mRNA that is likely to be the target of a microRNA. We have observed that insertion of this element into a luciferase reporter significantly reduces its expression. Importantly, a mutation in the target element that is predicted to compromise the binding of microRNAs abrogates the suppressive effect of the target element. Interestingly, this mutation has previously been identified as a rare polymorphism in African American populations. Thus, our overarching hypothesis is that the cannabinoid receptor is regulated by microRNAs and that variants in the cannabinoid receptor element may influence its expression and thus render the individual susceptible to drug addiction. In our first aim, we will confirm and extend our initial observations and identify the microRNA that mediates repression of the cannabinoid receptor. These studies will be aided by our experience in designing and using "Antagomir" reagents. Antagomirs are modified RNAs that can be introduced into cells and can potently down regulate the expression of specific microRNAs. In our second aim, we will examine whether any human variants in the cannabinoid receptor element correlate with susceptibility to addiction. To do this, we have established collaboration with Drs. Kranzler, Covault and Oncken who have access to DNA from control and affected patient populations.