Department of Neurosurgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Science, Beijing, China.
Eur Rev Med Pharmacol Sci. 2020 Apr;24(8):4420-4429. doi: 10.26355/eurrev_202004_21024.
To investigate the expression of circ_0072309 in patients with ischemic stroke (IS) and in LIFR humanized mice with middle cerebral artery occlusion (MCAO). Further, we explored the underlying mechanism of circ-0072309 in IS.
The content of circ-0072309 in serum of patients with IS (n = 70) was measured by qRT-PCR, and the ROC curve was analyzed. LIFR humanized mice were used to measure the content of circ-0072309 in ischemic hemisphere by qRT-PCR and the protein expression of cleaved-caspase-3, cleaved-caspase-8 were detected by Western blot. After that, the expression of miR-100 in serum of patients with IS and in ischemic hemisphere of MCAO mice were detected, and then, we analyzed the correlation between the expression of circ-0072309 and miR-100. The binding sites between circ-0072309 and miR-100 were predicted by online database. We detected whether cric-0072309 bind to miR-100 by Dual-Luciferase report in bEnd2. In addition, bEnd2 was treated with oxygen-glucose deprivation (OGD) to simulate injury of cerebral vascular after cerebral ischemia. After treated with miR-100 mimic or miR-100-inhibitor, we detected the cell survival and rate of cell apoptosis, and the content of cleaved-caspase-3 and caspase-8 protein. The target mRNA of miR-100 was predicted by bioinformatics analysis and analyzed by Dual-Luciferase. After treating bEnd2 with circ-0072309 and miR-100 mimic, we analyzed the cell survival and apoptosis to identify the potential regulatory mechanism.
The results of qRT-PCR showed that the expression of circ-0072309 was significantly decreased while the content of miR-100 was significantly increased in the serum of IS patients and in the ischemic hemisphere of MCAO mice. There was a negative correlation between the expression of circ-0072309 and miR-100. The results of Dual-Luciferase showed that circ-0072309 could directly bind to miR-100. After treating bEnd2 with OGD, miR-100-mimic caused a decrease rate of cell survival and an increased rate of apoptosis. Dual-Luciferase showed that miR-100 regulated cell survival and apoptosis by directly binding to mTOR. By comparing treated bEnd2 with circ-0072309, co-transfected bEnd2 with circ-0072309 and miR-100 reduced cell survival and increased apoptosis.
According to these results, this study revealed that the circ_0072309-miR-100-mTOR regulatory axis could alleviate IS, and it may be a potential target for the treatment of IS.
研究环状 RNA（circRNA）_0072309 在缺血性脑卒中（IS）患者和大脑中动脉闭塞（MCAO）模型中的表达。进一步探讨 circ-0072309 在 IS 中的作用机制。
采用实时荧光定量 PCR（qRT-PCR）检测 70 例 IS 患者血清中 circ-0072309 的含量，并绘制 ROC 曲线。采用 qRT-PCR 检测 MCAO 模型中缺血侧脑组织中 circ-0072309 的含量，Western blot 检测 cleaved-caspase-3 和 cleaved-caspase-8 蛋白的表达。检测 IS 患者血清和 MCAO 模型缺血侧脑组织中 miR-100 的表达，分析 circ-0072309 与 miR-100 的相关性。通过在线数据库预测 circ-0072309 与 miR-100 的结合位点。采用双荧光素酶报告基因检测验证 bEnd2 细胞中 circ-0072309 与 miR-100 的结合。此外，采用氧葡萄糖剥夺（OGD）模拟脑缺血后脑血管损伤，用 miR-100 模拟物或 miR-100 抑制剂处理 bEnd2 细胞，检测细胞存活率和细胞凋亡率，以及 cleaved-caspase-3 和 caspase-8 蛋白的含量。通过生物信息学分析预测 miR-100 的靶基因，并通过双荧光素酶报告基因进行验证。用 circ-0072309 和 miR-100 模拟物处理 bEnd2 细胞后，分析细胞存活和凋亡情况，以确定潜在的调控机制。
qRT-PCR 结果显示，IS 患者血清和 MCAO 模型缺血侧脑组织中 circ-0072309 的表达明显降低，miR-100 的含量明显升高。circ-0072309 与 miR-100 的表达呈负相关。双荧光素酶报告基因结果显示，circ-0072309 可与 miR-100 直接结合。用 OGD 处理 bEnd2 细胞后，miR-100 模拟物可降低细胞存活率，增加细胞凋亡率。双荧光素酶报告基因结果显示，miR-100 通过直接结合 mTOR 调节细胞存活和凋亡。与转染 circ-0072309 的 bEnd2 细胞比较，共转染 circ-0072309 和 miR-100 可降低细胞存活率，增加细胞凋亡率。
本研究揭示了 circ_0072309-miR-100-mTOR 调控轴可减轻 IS 损伤，可能成为 IS 治疗的潜在靶点。
