MicroRNA-181c Exacerbates Brain Injury in Acute Ischemic Stroke
Ma Qingfeng1,2, Zhao Haiping1,4, Tao Zhen1,4, Wang Rongliang1,4, Liu Ping1, Han Ziping1, Ma Shubei1, Luo Yumin1,3,4,*, Jia Jianping1,2,*
1Department of Neurology and Cerebrovascular Diseases Research Institute, Xuanwu Hospital of Capital Medical University 2Neurodegenerative Laboratory of Ministry of Education of the People’s Republic of China 3Beijing Institute for Brain Disorders 4Beijing Key Laboratory of Translational Medicine for Cerebrovascular Disease, Beijing, China
MicroRNA-181 (miR-181) is highly expressed in the brain, and downregulated in miRNA expression profiles of acute ischemic stroke patients. However, the roles of miR-181c in stroke are not known. The clinical relevance of miR-181c in acute stroke patients was evaluated by real-time PCR and correlation analyses. Proliferation and apoptosis of BV2 microglial cells and Neuro-2a cells cultured separately or together under oxidative stress or inflammation were assessed with the Cell Counting Kit-8 and by flow cytometry, respectively. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in C57/BL6 mice, and cerebral infarct volume, microglia activation, and expression of pro-apoptotic factors were evaluated by 2,3,5-triphenyl-2H-tetrazolium chloride staining, immunocytochemistry, and western blotting, respectively. Plasma levels of miR-181c were decreased in stroke patients relative to healthy individuals, and were positively correlated with neutrophil number and blood platelet count and negatively correlated with lymphocyte number. Lipopolysaccharide (LPS)/hydrogen peroxide (H2O2) treatment inhibited BV2 microglia proliferation without inducing apoptosis, while miR-181c reduced proliferation but increased the apoptosis of these cells with or without LPS/H2O2 treatment. LPS/H2O2 induced apoptosis in Neuro-2a cells co-cultured with BV2 cells, an effect that was potentiated by miR-181c. In the MCAO model, miR-181c agomir modestly increased infarct volume, markedly decreased microglia activation and B cell lymphoma-2 expression, and increased the levels of pro-apoptotic proteins in the ischemic brain. Our data indicate that miR-181c contributes to brain injury in acute ischemic stroke by promoting apoptosis of microglia and neurons via modulation of pro- and anti-apoptotic proteins.
Table 1 MiRNAs down- or upregulated in lymphocytes within 72 h of acute stroke.
Figure 1. Expression of miR-181c and miR181d in the plasma of acute stroke patients. Plasma levels of miR-181c (A) and miR-181d (B) in acute stroke patients and controls, as determined by semi-quantitative RT-PCR (n = 7 in the control group, n = 10 in the stroke group). U6 was used to normalize expression levels of target miRNAs in different samples.
Figure 2. Correlation between miR-181c level in plasma and NIHSS score (n = 7). (A), neutrophil number (n = 8) (B), lymphocyte number (n = 9) (C), and blood platelet count (n = 10) (D). P < 0.05 was used as a cutoff value.
Figure 3. MiR-181c agomir inhibits proliferation and induces apoptosis of BV2 microglial cells upon oxidative stress and inflammation. A) BV2 cell proliferation was assessed with Cell Counting Kit-8. B) BV2 cell apoptosis was determined by flow cytometry. Values represent mean ± SEM. *P < 0.05 vs. negative control group; #P < 0.05 vs. negative control + H2O2 or negative control + LPS group.
Figure 4. MiR-181c agomir promotes apoptosis of Neuro-2a cells upon oxidative stress and inflammation. A) Apoptotic cells were detected by flow cytometry. Second and fourth quadrants represent late and early apoptotic fractions, respectively. B) Early apoptosis of Neuro-2a cells. C) Late apoptosis of Neuro-2a cells. Values represent mean ± SEM. *P < 0.05 vs. negative control group; #P < 0.05 vs. negative control + H2O2 or negative control + LPS group.
Figure 5. MiR-181c agomir induces apoptosis of Neuro-2a cells co-cultured with BV2 microglial cells upon oxidative stress and inflammation. A) Apoptotic cells were detected by flow cytometry. Second and fourth quadrants represent late and early apoptotic fractions, respectively. B) Early apoptosis of Neuro-2a cells. C) Late apoptosis of Neuro-2a cells. *P < 0.05 vs. negative control group; #P < 0.05 vs. negative control + H2O2 or negative control + LPS group.
Figure 6. MiR-181c agomir exacerbates brain ischemia-reperfusion injury in an MCAO mouse model. A) Cerebral infarct volume was evaluated by TTC staining of coronal brain sections. B) MiR-181c agomir inhibited microglia activation in the ipsilateral cortex, as determined by immunocytochemistry. C) MiR-181c agomir inhibited the expression of the anti-apoptotic protein Bcl-2 in the ipsilateral cortex, as detected by immunocytochemistry. D) MiR-181c agomir increased the levels of the pro-apoptotic factors Bax and activated caspase-3 in the ipsilateral cortex, as detected by western blotting. Values represent mean ± SEM. *P < 0.05 vs. sham group, #P < 0.05 vs. MCAO + control group.
Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 380: 2095-2128.
Zhang L, Dong LY, Li YJ, Hong Z, Wei WS (2012). The microRNA miR-181c controls microglia-mediated neuronal apoptosis by suppressing tumor necrosis factor. J Neuroinflammation, 9: 211.
Zhang L, Li YJ, Wu XY, Hong Z, Wei WS (2015). MicroRNA-181c negatively regulates the inflammatory response in oxygen-glucose-deprived microglia by targeting Toll-like receptor 4. J Neurochem, 132:713-723.
Zhao H, Wang J, Gao L, Wang R, Liu X, Gao Z, et al. (2013). MiRNA-424 protects against permanent focal cerebral ischemia injury in mice involving suppressing microglia activation. Stroke, 44:1706-1713.
Liu P, Zhao H, Wang R, Wang P, Tao Z, Gao L, et al. (2015). MicroRNA-424 protects against focal cerebral ischemia and reperfusion injury in mice by suppressing oxidative stress. Stroke, 46:513-519.
Yin XM, Luo Y, Cao G, Bai L, Pei W, Kuharsky DK, et al. (2002). Bid-mediated mitochondrial pathway is critical to ischemic neuronal apoptosis and focal cerebral ischemia. J Biol Chem, 44: 42074-81.
Maestrini I, Strbian D, Gautier S, Haapaniemi E, Moulin S, Sairanen T, et al. (2015). Higher neutrophil counts before thrombolysis for cerebral ischemia predict worse outcomes. Neurology, 85:1408-1416.
Hyun S, Kwon S, Cho S, Park S, Jung W, Moon S, et al. (2015). Can the Neutrophil-to-Lymphocyte Ratio Appropriately Predict Carotid Artery Stenosis in Patients with Ischemic Stroke?-A Retrospective Study. J Stroke Cerebrovasc Dis, 24:2646-2651.
Wu C, Gong Y, Yuan J, Zhang W, Zhao G, Li H, et al. (2012). microRNA-181a represses ox-LDL-treated inflammatory response in dendritic cell by targeting c-Fos. J Lipid Res, 53: 2355-2363.
Wang XF, Shi ZM, Wang XR, Cao L, Wang YY, Zhang JX, et al. (2012). MiR-181d acts as a tumor suppressor in glioma by targeting K-ras and Bcl-2. J Cancer Res Clin Oncol, 138: 573-584.
Das S, Ferlito M, Kent OA, Fox-Talbot K, Wang R, Liu D, et al. (2012). Nuclear miRNA regulates the mitochondrial genome in the heart. Circ Res, 110:1596-1603.