Department of Neonatology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Department of Anesthesiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Brain Res Bull. 2023 Mar;194:90-99. doi: 10.1016/j.brainresbull.2023.01.013. Epub 2023 Jan 30.
Neonatal hypoxic ischemic encephalopathy (HIE) is a main factor of neonatal death and permanent neurologic deficit. This study sought to investigate the functional role of hsa_circ_0007706 (circ_0007706) in modulating neonatal HIE.
In vitro HIE cell model was established in hBMVECs under the condition of oxygen‑glucose deprivation/reperfusion (OGD/R) treatment. qRT-PCR analysis was utilized for detecting the level of circ_0007706, microRNA-579-3p (miR-579-3p) and TNF receptor-associated factor 6 (TRAF6). RNase R treatment and Oligo (dT) primers were employed to verify the features of circ_0007706, and nucleocytoplasmic separation was conducted for determining the location of circ_0007706. CCK-8 assay, EdU assay, and flow cytometry were carried out to measure cell proliferation and apoptosis, respectively. The protein expression of Bax, Bcl-2 and TRAF6 was detected using western blot. Meanwhile, the levels of the pro-inflammatory factors were determined via ELISA. SOD activity and MDA level were assessed via the respective kits. Besides, dual-luciferase reporter assay and RNA pull-down were used to identify the association between miR-579-3p and circ_0007706 or TRAF6.
Circ_0007706 was elevated in HIE newborns and OGD/R cell model. Knockdown of circ_0007706 greatly alleviated OGD/R-induced injury, inflammatory response and oxidative stress. We found that miR-579-3p was a direct target of circ_0007706, and miR-579-3p inhibitor could reverse the impact of circ_0007706 knockdown on OGD/R-caused cell damage in hBMVECs. In addition, miR-579-3p directly interacted with TRAF6, and the protective effects of miR-579-3p on OGD/R-induced injury in hBMVECs were harbored by TRAF6 overexpression. Our data indicated that circ_0007706 knockdown could downregulate the expression of TRAF6 by sponging miR-579-3p in OGD/R-treated hBMVECs.
This study demonstrated that circ_0007706 knockdown assuaged HIE-induced injury by decreasing TRAF6 expression via targeting miR-579-3p.
新生儿缺氧缺血性脑病（HIE）是新生儿死亡和永久性神经功能缺损的主要因素。本研究旨在探讨 hsa_circ_0007706（circ_0007706）在调节新生儿 HIE 中的功能作用。
在氧葡萄糖剥夺/再灌注（OGD/R）处理条件下，在 hBMVECs 中建立体外 HIE 细胞模型。利用 qRT-PCR 分析检测 circ_0007706、微小 RNA-579-3p（miR-579-3p）和肿瘤坏死因子受体相关因子 6（TRAF6）的水平。使用 RNase R 处理和 Oligo（dT）引物验证 circ_0007706 的特征，并进行核质分离以确定 circ_0007706 的位置。通过 CCK-8 测定、EdU 测定和流式细胞术分别测量细胞增殖和凋亡。使用 Western blot 检测 Bax、Bcl-2 和 TRAF6 的蛋白表达。同时，通过 ELISA 测定促炎因子的水平。通过相应的试剂盒评估 SOD 活性和 MDA 水平。此外，使用双荧光素酶报告基因检测和 RNA 下拉实验来鉴定 miR-579-3p 与 circ_0007706 或 TRAF6 之间的关联。
circ_0007706 在 HIE 新生儿和 OGD/R 细胞模型中升高。circ_0007706 敲低极大地减轻了 OGD/R 诱导的损伤、炎症反应和氧化应激。我们发现 miR-579-3p 是 circ_0007706 的直接靶标，并且 miR-579-3p 抑制剂可以逆转 circ_0007706 敲低对 hBMVECs 中 OGD/R 引起的细胞损伤的影响。此外，miR-579-3p 直接与 TRAF6 相互作用，并且 TRAF6 过表达可维持 miR-579-3p 对 hBMVECs 中 OGD/R 诱导损伤的保护作用。我们的数据表明，circ_0007706 敲低通过海绵吸附 miR-579-3p 下调 OGD/R 处理的 hBMVECs 中的 TRAF6 表达。
本研究表明，circ_0007706 敲低通过靶向 miR-579-3p 降低 TRAF6 表达来减轻 HIE 诱导的损伤。
