The decreased circular RNA hsa_circ_0072309 promotes cell apoptosis of ischemic stroke by sponging miR-100

Y. Zhao, J. Li, J. Li, L. Xu, W. Lian

Department of Neurosurgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Science, Beijing, China. lwpumch@hotmail.com

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• Neurology

OBJECTIVE: 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.
MATERIALS AND METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.

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