Fan Yun, Wei Lihong, Zhang Sanjun, Song Xueyun, Yang Jiaqing, He Xiaoxia, Zheng Xianzhao
Department of Neurology, Jiaozuo People's Hospital, No, 267, Jiefang Middle Road, Jiaozuo, 454002, Henan, China.
Neurochem Res. 2021 Apr;46(4):1019-1030. doi: 10.1007/s11064-020-03222-9. Epub 2021 Feb 2.
Cerebral ischemia-reperfusion (I/R) injury is the common symptom of ischemic stroke, which poses a heavy burden to human health. Long non-coding RNA (lncRNA) is indicated to be a critical regulator in cerebral ischemia. This study aims to reveal the effects of lncRNA small nucleolar RNA host gene 15 (SNHG15) on oxygen-glucose deprivation and reoxygenation (OGD/R)-induced neuron injury and underlying mechanism. The expression levels of SNHG15, microRNA-455-3p (miR-455-3p) and tumour protein p53 inducible nuclear protein 1 (TP53INP1) mRNA were determined by quantitative real time polymerase chain reaction in P12 cells. The protein levels of TP53INP1, cleaved caspase-3, caspase-3, B-cell lymphoma-2 and BCL2-associated x protein (Bax) were detected by western blot in P12 cells. Cell viability and apoptosis were revealed by cell counting kit-8 assay and flow cytometry analysis, respectively, in P12 cells. Caspase-3 activity, the levels of tumor necrosis factor-α and interleukin-1β and the production of reactive oxygen species (ROS) were severally determined by caspase-3 activity assay, Enzyme-linked immunosorbent assay and ROS detection assay in P12 cells. The binding relationship between miR-455-3p and SNHG15 or TP53INP1 was predicted by starbase online database, and identified by dual-luciferase reporter, RNA pull-down or RNA immunoprecipitation assay. SNHG15 expression and the mRNA and protein levels of TP53INP1 were dramatically upregulated, while miR-455-3p expression was apparently downregulated in OGD/R-induced PC12 cells. SNHG15 silencing hindered the effects of OGD/R treatment on cell viability, apoptosis, inflammation and oxidative in PC12 cells; however, these impacts were restored after miR-455-3p inhibitor transfection. Additionally, SNHG15 acted as a sponge of miR-455-3p and miR-455-3p bound to TP53INP1. SNHG15 contributed to OGD/R-induced neuron injury by regulating miR-455-3p/TP53INP1 axis, which provided a novel insight to study lncRNA-directed therapy in ischemia stoke.
脑缺血再灌注（I/R）损伤是缺血性中风的常见症状，给人类健康带来沉重负担。长链非编码RNA（lncRNA）被认为是脑缺血中的关键调节因子。本研究旨在揭示长链非编码RNA小核仁RNA宿主基因15（SNHG15）对氧糖剥夺复氧（OGD/R）诱导的神经元损伤的影响及其潜在机制。通过定量实时聚合酶链反应测定P12细胞中SNHG15、微小RNA-455-3p（miR-455-3p）和肿瘤蛋白p53诱导核蛋白1（TP53INP1）mRNA的表达水平。通过蛋白质印迹法检测P12细胞中TP53INP1、裂解的半胱天冬酶-3、半胱天冬酶-3、B细胞淋巴瘤-2和BCL2相关X蛋白（Bax）的蛋白水平。分别通过细胞计数试剂盒-8法和流式细胞术分析揭示P12细胞的细胞活力和凋亡情况。通过半胱天冬酶-3活性测定、酶联免疫吸附测定和活性氧（ROS）检测测定P12细胞中的半胱天冬酶-3活性、肿瘤坏死因子-α和白细胞介素-1β水平以及ROS的产生。通过starbase在线数据库预测miR-455-3p与SNHG15或TP53INP1之间的结合关系，并通过双荧光素酶报告基因、RNA下拉或RNA免疫沉淀测定进行鉴定。在OGD/R诱导的PC12细胞中，SNHG15的表达以及TP53INP1的mRNA和蛋白水平显著上调，而miR-455-3p的表达明显下调。SNHG15沉默可阻碍OGD/R处理对PC12细胞的细胞活力、凋亡、炎症和氧化的影响；然而，在转染miR-455-3p抑制剂后，这些影响得以恢复。此外，SNHG15作为miR-455-3p的海绵，且miR-455-3p与TP53INP1结合。SNHG15通过调节miR-455-3p/TP53INP1轴促进OGD/R诱导的神经元损伤，这为研究缺血性中风中lncRNA导向治疗提供了新的见解。
