Jiangsu Engineering Research Center for Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.
J Neurochem. 2013 Jan;124(1):15-25. doi: 10.1111/jnc.12057. Epub 2012 Nov 21.
In this study, we first characterized synaptosome microRNA (miRNA) profiles using microarray and qRT-PCR. MicroRNAs were detected in isolated synaptic vesicles, and Ago2 immunoprecipitation studies revealed an association between miRNAs and Ago2. Second, we found that miR-29a, miR-99a, and miR-125a were significantly elevated in synaptosome supernatants after depolarization. MiRNA secretion by the synaptosome was Ca(2+)-dependent and was inhibited by the exocytosis inhibitor, okadaic acid. Furthermore, application of nerve growth factor increased miRNA secretion without altering the spontaneous release of miRNAs. Conversely, kainic acid decreased miRNA secretion and enhanced the spontaneous release of miRNAs. These results indicate that synaptosomes could secrete miRNAs. Finally, synthesized miRNAs were taken up by synaptosomes, and the endocytosis inhibitor Dynasore blocked this process. After incubation with miR-125a, additional miR-125a was bound to Ago2 in the synaptosome, and expression of the miR-125a target gene (PSD95 mRNA) was decreased; these findings suggest that the ingested miRNAs were assembled in the RNA-induced silencing complex, resulting in the degradation of target mRNAs. To our knowledge, this is the first study that demonstrates the secretion of miRNAs by synaptosomes under physiological stimulation and demonstrates that secreted miRNAs might be functionally active after being taken up by the synaptic fraction via the endocytic pathway.
在这项研究中，我们首先使用微阵列和 qRT-PCR 对突触体 microRNA (miRNA) 谱进行了特征描述。在分离的突触小泡中检测到了 microRNAs，Ago2 免疫沉淀研究表明 microRNAs 与 Ago2 之间存在关联。其次，我们发现去极化后突触体上清液中 miR-29a、miR-99a 和 miR-125a 显著升高。突触体的 miRNA 分泌依赖于 Ca(2+)，并被细胞外泌体抑制剂 okadaic 酸抑制。此外，神经生长因子的应用增加了 miRNA 的分泌，而不改变 miRNAs 的自发释放。相反，红藻氨酸降低了 miRNA 的分泌并增强了 miRNAs 的自发释放。这些结果表明突触体可以分泌 miRNA。最后，合成的 miRNA 被突触体摄取，内吞抑制剂 Dynasore 阻断了这一过程。用 miR-125a 孵育后，突触体中的 Ago2 结合了额外的 miR-125a，miR-125a 靶基因 (PSD95 mRNA) 的表达减少；这些发现表明，摄取的 miRNAs 组装在 RNA 诱导的沉默复合物中，导致靶 mRNA 的降解。据我们所知，这是首次证明生理刺激下突触体分泌 miRNA，并证明通过内吞途径被突触部分摄取后，分泌的 miRNA 可能具有功能活性。
