Abstract

Objectives

Increasing evidence confirmed that miRNA plays a critical role in the occurrence and development of ischemic stroke. Here, the aim of this study was to examine the function and mechanisms of miR-195 in vascular endothelial cell apoptosis induced by oxygen–glucose deprivation (OGD).

Methods

This study intended to use OGD to simulate ischemia in vitro. The mRNA expression of miR-195, IKKα and NF-κB in human umbilical vein endothelial cells (HUVECs) were detected by RT-qPCR. The proliferation and apoptosis ability of HUVECs were evaluated using MTT assay, colony formation assay and flow cytometry, respectively. Western blot was applied to examine related protein expression. The interaction between miR-195 and IKKα was verified by dual-luciferase reporter gene assay.

Results

OGD significantly inhibited cell viability and induced cell apoptosis in HUVECs. Meanwhile, OGD treatment notably decreased the expression of miR-195, as well as enhanced NF-κB expression. Moreover, miR-195 directly interacted with IKKα and suppressed its expression. Mechanically, overexpression of miR-195 exhibited pro-proliferation and anti-apoptotic effect on HUVECs treated with OGD through targeting IKKα-mediated NF-κB pathway. At the molecular level, through suppressing IKKα/NF-κB pathway, miR-195 inhibited the expression of pro-apoptotic protein Bax and active caspase-3, but increased the expression of anti-apoptotic Bcl-2 in HUVECs.

Conclusions

Our finding uncovers the protective effect of miR-195 on the biological behavior of HUVECs via suppression of the NF-κB pathway induced by IKKα, which may provide a new potential strategy for ischemic stroke clinical treatment.

中文翻译：

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MiR-195 通过抑制 IKKα 介导的 NF-κB 通路减轻氧-葡萄糖剥夺/再灌注诱导的细胞凋亡

摘要

目标

越来越多的证据证实miRNA在缺血性脑卒中的发生和发展中起关键作用。在这里，本研究的目的是检查 miR-195 在氧-葡萄糖剥夺（OGD）诱导的血管内皮细胞凋亡中的功能和机制。

方法

本研究旨在使用OGD在体外模拟缺血。RT-qPCR检测人脐静脉内皮细胞(HUVECs)中miR-195、IKKα和NF-κB的mRNA表达。分别采用MTT法、集落形成法和流式细胞术评估HUVECs的增殖和凋亡能力。应用蛋白质印迹检查相关蛋白表达。miR-195 和 IKKα 之间的相互作用通过双荧光素酶报告基因测定验证。

结果

OGD显着抑制HUVECs中的细胞活力并诱导细胞凋亡。同时，OGD处理显着降低了miR-195的表达，并增强了NF-κB的表达。此外，miR-195 直接与 IKKα 相互作用并抑制其表达。在机械上，miR-195 的过表达通过靶向 IKKα 介导的 NF-κB 通路对 OGD 处理的 HUVEC 表现出促增殖和抗凋亡作用。在分子水平上，miR-195通过抑制IKKα/NF-κB通路，抑制促凋亡蛋白Bax和活性caspase-3的表达，但增加HUVECs中抗凋亡Bcl-2的表达。

结论

我们的发现揭示了 miR-195通过抑制 IKKα 诱导的 NF-κB 通路对 HUVECs 生物学行为的保护作用，这可能为缺血性卒中的临床治疗提供新的潜在策略。