Jimenez-Gonzalez Ada, García-Concejo Adrián, López-Benito Saray, Gonzalez-Nunez Verónica, Arévalo Juan Carlos, Rodriguez Raquel E
Institute of Neurosciences of Castilla y Leon (INCyL), University of Salamanca, Spain; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Department of Biochemistry and Molecular Biology, University of Salamanca, Spain; Institute of Neurosciences of Castilla y Leon (INCyL), University of Salamanca, Spain; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Biochim Biophys Acta. 2016 Jun;1860(6):1308-16. doi: 10.1016/j.bbagen.2016.03.001. Epub 2016 Mar 4.
Morphine is one of the first-line therapies for the treatment of pain despite its secondary effects. It modifies the expression of epigenetic factors like miRNAs. In the present study, we analyzed miR-212 and miR-132 and their implication in morphine effects in the zebrafish Central Nervous System (CNS) through the regulation of Bdnf expression.
We used control and knock-down zebrafish embryos to assess the effects of morphine in miRNAs 212/132 and mitotic or apoptotic cells by qPCR, immunohistochemistry and TUNEL assay, respectively. Bdnf and TrkB were studied by western blot and through a primary neuron culture. A luciferase assay was performed to confirm the binding of miRNAs 212/132 to mecp2.
Morphine exposure decreases miR-212 but upregulates miR-132, as wells as Bdnf and TrkB, and changes the localization of proliferative cells. However, Bdnf expression was downregulated when miRNAs 212/132 and oprm1 were knocked-down. Furthermore, we proved that these miRNAs inhibit mecp2 expression by binding to its mRNA sequence. The described effects were corroborated in a primary neuron culture from zebrafish embryos.
We propose a mechanism in which morphine alters the levels of miRNAs 212/132 increasing Bdnf expression through mecp2 inhibition. oprm1 is also directly involved in this regulation. The present work confirms a relationship between the opioid system and neurotrophins and shows a key role of miR-212 and miR-132 on morphine effects through the regulation of Bdnf pathway.
miRNAs 212/132 are novel regulators of morphine effects on CNS. Oprm1 controls the normal expression of Bdnf.
吗啡是治疗疼痛的一线疗法之一，尽管存在副作用。它会改变微小RNA（miRNA）等表观遗传因子的表达。在本研究中，我们通过调节脑源性神经营养因子（Bdnf）的表达，分析了miR - 212和miR - 132及其在吗啡对斑马鱼中枢神经系统（CNS）作用中的影响。
我们分别使用对照和敲低斑马鱼胚胎，通过定量聚合酶链反应（qPCR）、免疫组织化学和末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）分析，评估吗啡对miRNAs 212/132以及有丝分裂或凋亡细胞的影响。通过蛋白质免疫印迹法以及原代神经元培养研究Bdnf和酪氨酸激酶受体B（TrkB）。进行荧光素酶测定以确认miRNAs 212/132与甲基化CpG结合蛋白2（mecp2）的结合。
吗啡暴露会降低miR - 212，但上调miR - 132以及Bdnf和TrkB，并改变增殖细胞的定位。然而，当miRNAs 212/132和阿片受体μ1（oprm1）被敲低时，Bdnf表达下调。此外，我们证明这些miRNAs通过与其信使核糖核酸（mRNA）序列结合来抑制mecp2表达。在斑马鱼胚胎的原代神经元培养中证实了上述作用。
我们提出了一种机制，即吗啡通过抑制mecp2来改变miRNAs 212/132的水平，从而增加Bdnf表达。oprm1也直接参与这一调节。本研究证实了阿片系统与神经营养因子之间的关系，并表明miR - 212和miR - 132通过调节Bdnf途径在吗啡作用中起关键作用。
miRNAs 212/132是吗啡对中枢神经系统作用的新型调节因子。Oprm1控制Bdnf的正常表达。
