microRNA-219-5p和microRNA-365调控慢性吗啡耐受的分子机制研究

Morphine tolerance is a difficult problem that limits its clinical application. The molecular mechanism of morphine tolerance is still unclear. In our previous study, we used microRNA array(Exquon)and found that the expression levels of miR-219-5p and miR-365 of the lumbar spinal dorsal horn in morphine tolerance rats was significantly down-regulated than that of control group. In view of these miRNAs contained morphine tolerance target genes,we presume that miR-219-5p and miR-365 in the spinal dorsal horn or dorsal root ganglion (DRG)of L4-6 may participate in the process of morphine tolerance through the regulation of their corresponding target gene. In our study, we will observe dynamic expression changes of miR-219-5p and miR-365 during the process of morphine tolerance, and verify these miRNAs expression changes by stem-loop real time quantitative PCR. Moreover, we utilize the double-label immunofluorescence technique to locate the cell types of the lumbar spinal dorsal horn and DRG where the miRNAs express differently. We also combine bioinformatics and literature to predict and analyze the target genes that these miRNAs regulate, then we use lentiviral mediated miRNA overexpression vector or locked nucleic acid (LNA)-modified antisense oligonucleotide to up-regulate or down-regulate the expression of the miRNAs in vitro or spinal level, and verified this effects using fluorescent in situ hybridization and taqman assay, then observe the influence on the target genes and pain ethology of the morphine tolerance rats.The implementation of this project provides experimental evidence for the molecular mechanism that miR-219-5p and miR-365 regulate morphine tolerance in the spinal dorsal horn or DRG, and provides novel intervention targets for the prophylaxis and treatment of morphine tolerance.

吗啡耐受是限制其临床镇痛应用的一大医学难题。我们通过miRNA芯片发现,大鼠脊髓背角的miR-219和miR-365表达在吗啡耐受形成后明显下调，鉴于其靶基因中含有吗啡耐受相关基因，我们推测miR-219-5p和miR-365调控其相应靶基因参与吗啡耐受形成。为证实这一假说,本项目拟采用miRNA芯片、RT-qPCR及免疫荧光双标技术检测吗啡耐受形成过程中脊髓背角及背根神经节（DRG）的miR-219-5p和miR-365表达动态变化及其表达的细胞类型,结合生物信息学及荧光素酶报告基因筛选鉴定上述miRNAs调控的靶基因及相关信号通路,采用慢病毒载体及LNA（锁核酸）技术体外及脊髓水平上调及下调上述miRNAs的表达，观察其如何调控靶基因表达进而干预吗啡耐受形成。本项目为阐明miR-219-5p和miRNA-365参与吗啡耐受的机制提供实验依据，为吗啡耐受防治提供新的干预靶点。

吗啡耐受是限制其临床镇痛应用的一大医学难题。我们通过miRNA芯片发现,大鼠脊髓背角的miR-219和miR-365表达在吗啡耐受形成后明显下调，鉴于其靶基因中含有吗啡耐受相关基因，我们推测miR-219-5p和miR-365调控其相应靶基因参与吗啡耐受形成。为证实这一假说,本项目拟采用miRNA芯片、RT-qPCR及免疫荧光双标技术检测吗啡耐受形成过程中脊髓背角及背根神经节（DRG）的miR-219-5p和miR-365表达动态变化及其表达的细胞类型,结合生物信息学及荧光素酶报告基因筛选鉴定上述miRNAs调控的靶基因及相关信号通路,采用慢病毒载体及LNA（锁核酸）技术体外及脊髓水平上调及下调上述miRNAs的表达，观察其如何调控靶基因表达进而干预吗啡耐受形成。本项目研究发现大鼠脊髓背角miR-365靶向β-arrestin 2 可以预防及逆转吗啡耐受形成。本项目发现大鼠脊髓背角的miR-219-5p通过作用CaMKIIγ/NMDA通路减轻吗啡耐受。本项目为阐明miR-219-5p和miRNA-365参与吗啡耐受的机制提供实验依据，为吗啡耐受防治提供新的干预靶点。