伏隔核微小RNA与靶基因CREB和AKT交互作用调控海洛因复吸

miRNAs as a class of gene expression modulators acting at the post-transcriptional level induce messenger RNA degradation and repress mRNA translation in a sequence-specific manner. Previous studies in our Lab.have shown that CREB or Akt signaling in the nucleus accumbens plays a negative or positive component in regulating heroin seeking behavior induced by cues after withdrawal. However，the miRNAs profiles target specifically on CREB and Akt in the accumbens nucleus and their post-transcriptional regulation mechanism underlying heroin seeking behavior are still unclear. The aim of project first is to identify the role of Akt and CREB isoforms of the accumbens nuclear in the heroin seeking after withdrawal by using Small interfering RNA. Based on the miRNA sequencing and bioinformatics prediction, the up- or down-regulated miRNA profiling which matches to CREB and Akt mRNA sequence is determined in the accumbens nucleus. Each candidate miRNA targets on Akt and CREB will be verified by the qPCR and Dual-Luciferase® Reporter Assay System.Then,the role of individually validated miRNA in heroin seeking behavior will be observed by knockdown and over-expression of this miRNA in vivo. Meanwhile, the regulative effect of miRNA on mRNA or protein expression of Akt and CREB and its downstream signaling molecules will be analyzed after test of heroin seeking. Only the miRNA regulating typically heroin seeking and expression of Akt or CREB is selected to assay its effect on neuronal plasticity after knockdown or over-expression in the nucleus accumbens. The project will demonstrate that Akt and CREB signaling play a key role in the heroin seeking behavior, and provide the evidence that the dynamic balance of miRNA and its target gene Akt and CREB contributes to heroin seeking after withdrawal, and also provide the candidate target to develop the potential therapy for treatment of heroin relapse and addiction.

miRNA能识别降解靶mRNA或抑制其翻译，在转录后水平调控基因表达。申请人前期研究发现伏隔核CREB 和AKT可负性和正性调节戒断后海洛因复吸，但海洛因复吸时伏隔核miRNA对CREB和AKT转录后调控和机制尚不清楚。本项目应用在体干扰技术查明伏隔核AKT和CREB亚型在海洛因复吸中的作用；并结合miRNA测序和生物信息学预测，确定伏隔核CREB 和AKT匹配的上调或下调miRNA谱，通过qPCR、细胞双转实验逐个验证miRNA对AKT和CREB的调节；通过在体敲减和过表达方式观察miRNA对海洛因复吸行为的调节，同时查明miRNA对AKT和CREB的mRNA、蛋白表达和下游信号分子的影响，并有选择地分析miRNA对伏隔核神经元可塑性的影响。该项目将明确靶基因和微小RNA的动态平衡在海洛因复吸中的关键作用和调控方式，有助于对海洛因复吸机制的认识，为开发靶向治疗海洛因成瘾提供实验依据。

海洛因成瘾的本质特征是持续心理渴求和复吸，本项目聚焦伏隔核CREB 和AKT对戒断后海洛因复吸的作用；利用miRNA测序等确定伏隔核miRNA谱，通过在体敲减和过表达观察miRNA对海洛因复吸行为的调节；查明miRNA对潜在靶基因分子的影响，该项目将探索靶基因和微小RNA动态平衡在复吸中的作用，有助于对海洛因复吸机制认识。我们发现随着大鼠戒断时间延长，觅药行为增加，伏隔核中p-Akt的表达增加；伏隔核注射PI3K抑制剂阻断戒断后线索诱导的海洛因觅药行为；线索诱导的海洛因觅药行为可被伏隔核Akt1的下调所抑制，而下调β-arrestin2不起作用，结果证明了PI3K/Akt通路可能参与海洛因复吸和成瘾的过程。经Western blot及RT-PCR检测后发现戒断14天后伏隔核AKT3 的mRNA和蛋白表达显著增高，而miR-382表达减少，miR-382通过与AKT3 mRNA碱基互补配对抑制AKT3基因的表达。在体升高伏隔核miR-382抑制线索诱导的大鼠海洛因复吸行为以及对AKT3表达的影响。通过RNA干扰敲减伏隔核Akt3可以减少AKT3 的mRNA和蛋白表达，同时抑制线索诱导海洛因复吸行为；通过建立敲除Akt3小鼠， AKT3敲除小鼠的海洛因自身给药行为以及线索诱导的觅药行为均明显下降。结果首次证明伏隔核AKT3参与线索诱导海洛因复吸，伏隔核AKT3受到miR-382的靶向调节。通过激活孤啡肽受体提高伏隔核CREB的表达和磷酸化水平，可以呈剂量依赖方式减少药物奖赏、奖赏动机以及复吸行为，提示CREB分子海洛因复吸行为和成瘾中的关键作用。我们发现miR-181a可能通过调控MeCP2参与海洛因复吸行为；伏隔核miR-206通过调控GABBR1表达发挥海洛因复吸保护作用；伏隔核miR-21-5p对海洛因复吸具有负性调节作用，而miR-320-3p具有正性调节作用， miR-21-5p和miR-320-3p可能通过动态调控ERK2靶基因的表达发挥作用。外周血浆hsa-miR-181a、miR-320a和let-7b水平在海洛因成瘾病人中显著增加，ROC曲线下面积分别为0.78、0.73和0.74；灵敏度为87%、65%和61%；特异度为55%、79%和87%。这些循环miRNAs对海洛因成瘾具有重要的诊断价值，有望成为诊断海洛因成瘾的新的无创性生物标志物。