Institute of Neuroanatomy, Medical Faculty, RWTH Aachen University, 52074, Aachen, Germany.
Department of Neurology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
Mol Neurobiol. 2021 Apr;58(4):1535-1549. doi: 10.1007/s12035-020-02210-y. Epub 2020 Nov 19.
MicroRNAs (miRNA) are small noncoding sequences that control apoptosis, proliferation, and neuroinflammatory pathways in microglia cells. The expression of distinct miRNAs is altered after ischemia in the brain. Only minor information is available about the biogenesis and maturation of miRNAs after ischemia. We aimed at examining the impact of oxygen-glucose deprivation (OGD) and hydrogen peroxide (HO)-induced stress on the expression of miRNA regulating proteins such as DROSHA, DGCR8, XPO5, DICER, TARBP2, and AGO2 in the cultured human microglial cell line HMC-3 (human microglial cell line clone 3). OGD duration of 2.5 h or HO stimulation at a concentration of 100 μM for 24 h resulted in a marked increase of the hypoxia sensor hypoxia-inducible factor1-α in HMC-3 cells. These treatments also led to an upregulation of DROSHA, DICER1, and AGO2 detected by semiquantitative real-time PCR (qrtPCR). XPO5 and TARBP2 were only upregulated after stimulation with HO, while DGCR8 responded only to OGD. We found elevated DICER1, DROSHA, and AGO2 protein levels by western blot and immunohistochemistry staining. Interestingly, the latter also exposed a colocalization of AGO2 with stress granules (G3BP1) after OGD. Our data indicate that DICER, DROSHA, and AGO2 are induced in microglial cells under hypoxia-like conditions. It might be speculated that their inductions might increase the miRNA synthesis rate. Future studies should investigate this correlation to determine which miRNAs are preferably expressed by microglia cells after ischemia and which functions they could exert.
微小 RNA（miRNA）是一种小的非编码序列，可控制小胶质细胞细胞中的细胞凋亡、增殖和神经炎症途径。在大脑中发生缺血后，不同的 miRNA 的表达会发生改变。关于缺血后 miRNA 的生物发生和成熟，仅有少量信息。我们的目的是研究氧葡萄糖剥夺（OGD）和过氧化氢（HO）诱导的应激对调节 miRNA 的蛋白质（如 DROSHA、DGCR8、XPO5、DICER、TARBP2 和 AGO2）在培养的人小胶质细胞系 HMC-3（人小胶质细胞系克隆 3）中的表达的影响。2.5 小时的 OGD 或 100 μM 的 HO 刺激 24 小时导致 HMC-3 细胞中缺氧传感器缺氧诱导因子 1-α（HIF-1-α）的显著增加。这些处理还导致 DROSHA、DICER1 和 AGO2 的上调，这可以通过半定量实时 PCR（qrtPCR）检测到。XPO5 和 TARBP2 仅在受到 HO 刺激后上调，而 DGCR8 仅对 OGD 有反应。我们通过 Western blot 和免疫组织化学染色发现 DICER1、DROSHA 和 AGO2 的蛋白水平升高。有趣的是，后者还显示出 AGO2 在 OGD 后与应激颗粒（G3BP1）的共定位。我们的数据表明，在缺氧样条件下，小胶质细胞中诱导了 DICER、DROSHA 和 AGO2。可以推测，它们的诱导可能会增加 miRNA 的合成速率。未来的研究应该调查这种相关性，以确定缺血后小胶质细胞细胞优先表达哪些 miRNA，以及它们可以发挥哪些功能。
