长链非编码 RNA-MIAT 通过上调 REDD1 促进缺血性脑卒中神经元细胞自噬和凋亡。
LncRNA-MIAT promotes neural cell autophagy and apoptosis in ischemic stroke by up-regulating REDD1.
机构信息
Department of Internal Neurology, Huaihe Hospital of Henan University, Kaifeng 475000, China.
Department of Internal Neurology, Huaihe Hospital of Henan University, Kaifeng 475000, China.
出版信息
Brain Res. 2021 Jul 15;1763:147436. doi: 10.1016/j.brainres.2021.147436. Epub 2021 Mar 18.
BACKGROUND
Ischemic stroke (IS) accounts for 80% of stroke incidence, which has an impact on the life quality of patients. Long non-coding RNA (LncRNA), a class of non-coding transcripts greater than 200 nucleotidesin length, has been extensively studied in cerebrovascular diseases. Myocardial infarction associated transcript (MIAT) is highly expressed in nervous system. Therefore this study aims to explore the role of LncRNA MIAT in IS and to clarify its underlying mechanism, providing therapeutic value for the treatment of IS.
METHODS
The neurological function of rats was evaluated by neurological deficit score. Triphenyltetrazolium chloride (TTC) staining was used to detect infarct area in brain tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of MIAT. Western blotting was used to detect the expressions of REDD1, p-mTOR, autophagy-related proteins LC3 and p62, and apoptotic-related proteins Bax, cleaved-caspase3, Bcl-2. Flow cytometry was applied to examine neuronal cell apoptosis. RNA pull-down and RIP assay was used to verify the binding of MIAT and REDD1. The level of REDD1 ubiquitination was detected by ubiquitination and Co-immunoprecipitation (Co-IP) assay.
RESULTS
The expressions of MIAT and REDD1 were increased in IS rats and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced PC12 cell injury. After interference with si-MIAT, the results of flow cytometry showed that the rate of apoptosis was reduced. Western blotting results showed that the expression of LC3II/LC3I, Bax, and cleaved-caspase3 was decreased, while the expression of p-mTOR, p62, and Bcl-2 was increased. RNA pull-down and RIP assay found the binding relationship between MIAT and REDD1, and interference with si-MIAT down-regulated the expression of REDD1. The level of REDD1 ubiquitination was increased and the expression of REDD1 was decreased after interference with si-MIAT in PC12 cells. Co-IP results showed that interference with si-MIAT enhanced the binding ability of CUL4A-DDB1 and REDD1.
CONCLUSION
Altogether, MIAT promotes autophagy and apoptosis of neural cells and aggravates IS by up-regulating the expression of REDD1.
背景
缺血性脑卒中（IS）约占脑卒中发病率的 80%，对患者的生活质量产生影响。长链非编码 RNA（LncRNA）是一类长度大于 200 个核苷酸的非编码转录本，在脑血管疾病中得到了广泛的研究。心肌梗塞相关转录物（MIAT）在神经系统中高度表达。因此，本研究旨在探讨 LncRNA MIAT 在 IS 中的作用，并阐明其潜在机制，为 IS 的治疗提供治疗价值。
方法
通过神经功能缺损评分评估大鼠的神经功能。三苯基四唑氯（TTC）染色用于检测脑组织中的梗死面积。采用定量实时聚合酶链反应（qRT-PCR）检测 MIAT 的表达。Western blot 用于检测 REDD1、p-mTOR、自噬相关蛋白 LC3 和 p62 以及凋亡相关蛋白 Bax、cleaved-caspase3、Bcl-2 的表达。流式细胞术用于检测神经元细胞凋亡。RNA 下拉和 RIP 测定用于验证 MIAT 和 REDD1 的结合。通过泛素化和免疫共沉淀（Co-IP）测定检测 REDD1 泛素化水平。
结果
IS 大鼠和氧葡萄糖剥夺/复氧（OGD/R）诱导的 PC12 细胞损伤中 MIAT 和 REDD1 的表达增加。干扰 si-MIAT 后，流式细胞术结果显示细胞凋亡率降低。Western blot 结果显示 LC3II/LC3I、Bax 和 cleaved-caspase3 的表达减少，而 p-mTOR、p62 和 Bcl-2 的表达增加。RNA 下拉和 RIP 测定发现 MIAT 和 REDD1 之间存在结合关系，干扰 si-MIAT 可下调 REDD1 的表达。干扰 si-MIAT 后，PC12 细胞中 REDD1 的泛素化水平增加，表达减少。Co-IP 结果显示，干扰 si-MIAT 增强了 CUL4A-DDB1 和 REDD1 的结合能力。
结论
综上所述，MIAT 通过上调 REDD1 的表达促进神经细胞的自噬和凋亡，加重 IS。
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