Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China; Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, Sichuan University, 610041 Chengdu, Sichuan, China.
Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.
Neurobiol Dis. 2014 Mar;63:184-93. doi: 10.1016/j.nbd.2013.11.023. Epub 2013 Dec 10.
Human growth transformation dependent protein (HGTD-P) is a newly identified protein that promotes neuronal apoptosis in hypoxia-ischemia brain damage (HIBD) in neonatal rats. However, the mechanisms regulating HGTD-P expression are not clear. Here we describe microRNAs targeted to HGTD-P and examine their effects on regulating neuronal apoptosis in HIBD. We use samples from cultured neurons after oxygen-glucose deprivation (OGD) and postnatal day 10 rat brains after hypoxia-ischemia (HI). RT-PCR, Western blotting, and immunostaining are used to detect the expression of HGTD-P and cleaved caspase 3, as well as real-time PCR detects microRNA expression. MicroRNA agomir is used to inhibit the expression of HGTD-P, and DAPI, TUNEL, and TTC staining are employed to detect cell apoptosis and brain damage. Moreover, in vitro processing assay is used to examine the mechanism by which HI down-regulates miR-139-5p expression. We found that miR-139-5p is down-regulated in neurons and rat brains after HI treatment. The expression pattern of miR-139-5p correlates inversely with that of HGTD-P. Furthermore, miR-139-5p agomir inhibits neuronal apoptosis and attenuates HIBD, which is concurrent with down-regulation of HGTD-P. Moreover, pre-miR-139 processing activity decreases in extracts from OGD neurons, and OGD neuronal extracts attenuates the processing of pre-miR-139 by Dicer. In conclusion, HI induces inhibitors which block the processing step of pre-miR-139, resulting in the down-regulation of mature miR-139-5p. The down-regulation of miR-139-5p plays a critical role in the up-regulation of HGTD-P expression. MiR-139-5p agomir attenuates brain damage when used 12h after HI, providing a longer therapeutic window than anti-apoptosis compounds currently available.
人类生长转化依赖蛋白（HGTD-P）是一种新鉴定的蛋白，可促进新生大鼠缺氧缺血性脑损伤（HIBD）中的神经元凋亡。然而，调节 HGTD-P 表达的机制尚不清楚。在这里，我们描述了靶向 HGTD-P 的 microRNAs，并研究了它们对调节 HIBD 中神经元凋亡的作用。我们使用氧葡萄糖剥夺（OGD）后培养的神经元样本和缺氧缺血（HI）后 10 天大鼠脑样本。使用 RT-PCR、Western blot 和免疫染色检测 HGTD-P 和 cleaved caspase 3 的表达，实时 PCR 检测 microRNA 表达。使用 microRNA agomir 抑制 HGTD-P 的表达，并用 DAPI、TUNEL 和 TTC 染色检测细胞凋亡和脑损伤。此外，体外处理测定用于检查 HI 下调 miR-139-5p 表达的机制。我们发现，miR-139-5p 在 HI 处理后的神经元和大鼠脑中下调。miR-139-5p 的表达模式与 HGTD-P 呈负相关。此外，miR-139-5p agomir 抑制神经元凋亡并减轻 HIBD，同时下调 HGTD-P。此外，OGD 神经元提取物中的 pre-miR-139 加工活性降低，并且 OGD 神经元提取物减弱 Dicer 对 pre-miR-139 的加工。总之，HI 诱导抑制剂阻止 pre-miR-139 的加工步骤，导致成熟 miR-139-5p 的下调。miR-139-5p 的下调在 HGTD-P 表达的上调中起关键作用。miR-139-5p agomir 在 HI 后 12 小时使用可减轻脑损伤，提供比目前可用的抗凋亡化合物更长的治疗窗口。
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