| 鲁传豪,孙应辉,李肖亮,袁晨阳,曹建华,汪仁聪.miR-96-5p靶向FOXO1调节脑缺血再灌注损伤机制[J].,2023,(12):2221-2228 |
| miR-96-5p靶向FOXO1调节脑缺血再灌注损伤机制 |
| miR-96-5p Regulates the Mechanism of Cerebral Ischemia Reperfusion Injury by Targeting FOXO1 |
| 投稿时间：2023-02-23 修订日期：2023-03-18 |
| DOI：10.13241/j.cnki.pmb.2023.12.004 |
| 中文关键词: 缺血性脑卒中 脑缺血再灌注损伤 miR-96-5p Forkhead box O1 氧化应激 |
| 英文关键词: Ischemic stroke Cerebral ischemia-reperfusion injury miR-96-5p Forkhead box O1 Oxidative stress |
| 基金项目:陕西省重点研发计划-社会发展领域（2019SF-046） |
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| 中文摘要: |
| 摘要 目的：探究miR-96-5p在脑缺血再灌注损伤（CIRI）中的作用及机制。方法：通过qRT-PCR检测36例确诊的缺血性脑卒中患者（IS患者组）和30例健康体检者（健康组）的血清miR-96-5p水平。将PC12细胞分为5组：对照组、NC-ag组、miR-96-5p-ag组、NC-an组、miR-96-5p-an组。采用Lipofectamine 2000对PC12细胞进行转染，通过qRT-PCR验证转染效率。将PC12细胞分为6组：对照组、氧糖剥夺/复氧复糖（OGD/R）组、OGD/R+NC-ag组、OGD/R+miR-ag组、OGD/R+NC-an组、OGD/R+miR-an组。根据分组对PC12细胞进行OGD/R处理和转染。通过MTT法检测PC12细胞活力，TUNEL法检测PC12细胞凋亡。采用改良Longa法建立大鼠CIRI模型，然后将大鼠分为假手术组、CIRI组、CIRI+NC-an组和CIRI+miR-an组。假手术组和CIRI组大鼠尾静脉注射生理盐水，CIRI+NC-an组和CIRI+miR-an组大鼠分别尾静脉注射NC-antagomir和miR-96-5p-antagomir。然后检测各组大鼠的神经功能评分、脑梗死体积和脑组织细胞凋亡情况。按照试剂盒说明书测定PC12细胞和大鼠脑组织中MDA、SOD和GSH-Px的含量。通过qRT-PCR检测PC12细胞和大鼠脑组织miR-96-5p和Forkhead box O1（FOXO1） mRNA水平，通过Western blot检测FOXO1、Ac-FOXO1、Bax和Bcl-2的蛋白表达水平。结果：与Health组比较，IS组患者的血清miR-96-5p水平显著升高（P<0.001）。与对照组和NC-ag组比较，miR-96-5p-ag组的miR-96-5p水平升高，FOXO1的mRNA和蛋白表达水平均降低，FOXO1的乙酰化水平升高（P<0.05）。与对照组和NC-an组比较，miR-96-5p-an组的miR-96-5p水平降低，FOXO1的mRNA和蛋白表达水平均升高，FOXO1的乙酰化水平降低（P<0.05）。与OGD/R组和OGD/R+NC-an组比较，OGD/R+miR-an组的相对细胞活力升高，TUNEL阳性率降低，Bax的蛋白相对表达量降低，Bcl-2的蛋白相对表达量升高，MDA水平降低，SOD和GSH-Px水平升高，miR-96-5p水平降低，FOXO1的mRNA和蛋白表达水平升高，FOXO1的乙酰化水平降低（P<0.05）。与CIRI组和CIRI+NC-an组比较，CIRI+miR-an组大鼠的神经功能评分和脑梗死体积降低，TUNEL阳性率降低，Bax的蛋白相对表达量降低，Bcl-2的蛋白相对表达量升高，MDA水平降低，SOD和GSH-Px水平升高，miR-96-5p水平降低，FOXO1的mRNA和蛋白表达水平升高，FOXO1的乙酰化水平降低（P<0.05）。结论：miR-96-5p在CIRI发生过程中表达上调，而下调miR-96-5p表达可能通过负调控FOXO1以减轻CIRI程度。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the role and mechanism of miR-96-5p in cerebral ischemia reperfusion injury (CIRI). Methods: Serum mRNA level of miR-96-5p was detected in 36 patients diagnosed with ischemic stroke (IS group) and 30 healthy subjects (Health group) by qRT-PCR. PC12 cells were divided into 5 groups: Control group, NC-ag group, miR-96-5p-ag group, NC-an group, miR-96-5p-an group. PC12 cells were transfected with Lipofectamine 2000. The transfection efficiency was verified by qRT-PCR. PC12 cells were divided into the following 6 groups: Control group, OGD/R group, OGD/R+NC-agomir group, OGD/R+miR-96-5p- agomir group, OGD/R+NC-antagomir group, OGD/R+miR-96-5p-antagomir group. PC12 cells were treated with OGD/R and transfected according to the group scheme. PC12 cell viability was detected by MTT and apoptosis was detected by TUNEL. The modified Longa method was used to establish the CIRI model of rats, and the rats were divided into Sham group, CIRI group, CIRI+NC-an group and CIRI+miR-an group. Rats in Sham group and CIRI group were injected normal saline through tail vein. CIRI+NC-an group and CIRI+miR-an group were injected NC-antagomir and miR-96-5p-antagomir, respectively. Then the neurological function score, cerebral infarct volume and apoptosis of brain tissue of rats in each group were detected. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in PC12 cells and rat brain were determined according to the kit instructions. levels of miR-96-5p and Forkhead box O1 (FOXO1) mRNA in PC12 cells and rat brain tissues were detected by qRT-PCR, and protein expression levels of FOXO1, Ac-FOXO1, Bax and Bcl-2 were detected by Western blot. Results: Compared with the Health group, the serum miR-96-5p mRNA level in the IS group was significantly increased (P<0.001). Compared with Control group and NC-ag group, the miR-96-5p level in miR-96-5p-ag group was increased, the mRNA and protein expression levels of FOXO1 were decreased, and the acetylation level of FOXO1 was increased (P<0.05). Compared with Control group and NC-an group, the miR-96-5p level in miR-96-5p-an group was decreased, the mRNA and protein expression levels of FOXO1 were increased, and the acetylation level of FOXO1 was decreased (P<0.05). Compared with OGD/R group and OGD/R+NC-an group, the relative cell activity of OGD/R+miR-an group was increased, the positive rate of TUNEL was decreased, the relative expression level of Bax protein was decreased, the relative expression level of Bcl -2 protein was increased, the level of MDA was decreased. SOD and GSH-Px levels were increased, miR-96-5p levels were decreased, FOXO1 mRNA and protein expression levels were increased, and FOXO1 acetylation level was decreased (P<0.05). Compared with CIRI group and CIRI+NC-an group, the m nerve function score and cerebral infarction volume of CIRI+miR-an group were decreased, the positive rate of TUNEL was decreased, the relative expression level of Bax protein was decreased, the relative expression level of Bcl -2 protein was increased, the level of MDA was decreased. SOD and GSH-Px levels were increased, miR-96-5p levels were decreased, FOXO1 mRNA and protein expression levels were increased, and FOXO1 acetylation level was decreased (P<0.05). Conclusion: miR-96-5p is up-regulated in the process of CIRI occurrence, and down-regulation of miR-96-5p may alleviate CIRI through negative regulation of FOXO1. |
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