Inhibition of microRNA-29b suppresses oxidative stress and reduces apoptosis in ischemic stroke

Abstract

MicroRNAs (miRNAs) regulate protein expression by antagonizing the translation of mRNAs and are effective regulators of normal nervous system development, function, and disease. MicroRNA-29b (miR-29b) plays a broad and critical role in brain homeostasis. In this study, we tested the function of miR-29b in animal and cell models by inhibiting miR-29b expression. Mouse models of middle cerebral artery occlusion were established using the modified Zea-Longa suture method. Prior to modeling, 50 nmol/kg miR-29b antagomir was injected via the tail vein. MiR-29b expression was found to be abnormally increased in ischemic brain tissue. The inhibition of miR-29b expression decreased the neurological function score and reduced the cerebral infarction volume and cell apoptosis. In addition, the inhibition of miR-29b significantly decreased the malondialdehyde level, increased superoxide dismutase activity, and Bcl-2 expression, and inhibited Bax and Caspase3 expression. PC12 cells were treated with glutamate for 12 hours to establish in vitro cell models of ischemic stroke and then treated with the miR-29 antagomir for 48 hours. The results revealed that miR-29b inhibition in PC12 cells increased Bcl-2 expression and inhibited cell apoptosis and oxidative damage. These findings suggest that the inhibition of miR-29b inhibits oxidative stress and cell apoptosis in ischemic stroke, producing therapeutic effects in ischemic stroke. This study was approved by the Laboratory Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University (approval No. 201709276S) on September 27, 2017.

Keywords: Akt; PI3K; apoptosis; cerebral artery occlusion; ischemic stroke; malondialdehyde; microRNA-29b; oxidative stress; superoxide dismutase.

Figure 1

MiR-29b expression increases in cerebral ischemia mouse brain tissue and glutamate-treated PC12 cells. (A) Real-time polymerase chain reaction detection of miR-29b levels in the whole brain of MCAO mice after 24 hours of reperfusion (n= 6 in each group). (B) Glutamate-induced miR-29b expression in PC12 cells. PC12 cells were seeded on 12-well plates in complete medium for 24 hours prior to 12-hour exposure to glutamate at different concentrations (0, 12.5, 25, 50, 100 mM). Each experiment was repeated three times. Data are represented as the mean ± SD. *P< 0.05 (Student's t-test). MCAO: Middle cerebral artery occlusion; miR-29b: microRNA-29b.

Figure 2

MiR-29b increases glutamate-induced PC12 cell apoptosis. PC12 cells were pretreated with miR-29b mimic, miR-29b inhibitor, and respective negative controls for 48 hours, followed by incubation with glutamate (12.5 mM) for 12 hours. (A, B) Flow cytometry detection of the percentage of PC12 cell apoptosis. All data are expressed as the mean ± SD. *P< 0.05 (Student's t-test). (C) Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay of apoptotic PC12 cells (original magnification, 200×). The brightness of green fluorescence represents the degree of apoptosis. MiR-29b mimics induced apoptosis in PC12 cells, and miR-29b inhibitors decreased PC12 cell apoptosis. Each experiment was repeated three times. ctrl: Control; miR-29b: microRNA-29b.

Figure 3

MiR-29b decreases SOD activity and increases MDA content in PC12 cells. PC12 cells were pretreated with miR-29b mimic, miR-29b inhibitor, and the respective negative control for 48 hours, followed by incubation with glutamate (12.5 mM) for 12 hours. (A) MDA content in PC12 cells. (B) SOD activity in PC12 cells. (C) SOD-1 protein bands in PC12 cells, as determined by western blot assay. (D) Quantitative results of SOD-1 protein expression, which was normalized against β-actin. Values are represented as the mean ± SD. *P< 0.05 (Student's t-test). Each experiment was repeated three times. ctrl: Control; MDA: malondialdehyde; miR-29b: microRNA-29b; SOD: superoxide dismutase.

Figure 4

MiR-29b antagomir ameliorates the neurological deficits in cerebral ischemia model mice Mice were pre-treated with miR-29b antagomir control for 24 hours of reperfusion via the tail vein and then subjected to 2 hours of MCAO. (A) MiR-29b levels in the whole brain were decreased in the MCAO + miR-29b antagomir group compared with the MCAO + antagomir control group. (B) Neurological scores were assessed using a modified 6-point scoring system after 24 hours of reperfusion. (C) Representative pictures of 2,3,5-triphenyltetrazolium chloride staining showing the cerebral infarct size in brain sections. The infracted tissue remained white, whereas normal brain tissues were stained red. (D) Quantification of infarct sizes. Data are expressed as the mean ± SD (n= 6 in each group). *P< 0.05 (Student's t-test). MCAO: Middle cerebral artery occlusion; miR-29b: microRNA-29b.

Figure 5

MiR-29b antagomir inhibits apoptosis in cerebral ischemia model mice. (A) Caspase3 immunopositivity (red, stained by fluorescein) in mouse brain tissue (original magnification, ×200). The immunopositivity of Caspase3 protein in the brain tissue of the MCAO group was significantly higher than that of the control group, and that of the MCAO + miR-29b antagomir group was significantly decreased. The green arrow indicates the positive expression area. (B, C) Flow cytometry detection of the percentage of apoptotic cells in mouse brain tissue. (D) Bands of caspase3 protein expression in mouse brain tissue detected by western blot assay. (E) Quantitative results showing caspase3 protein expression, which was normalized to β-actin. Values are represented as the mean ± SD (n= 6 in each group). *P< 0.05 (Student's t-test). DAPI: 4′,6-Diamidino-2-phenylindole; miR-29b: microRNA-29b.

Figure 6

MiR-29b antagomir upregulates antioxidant systems in mouse brain tissues following cerebral ischemia. (A, B) Quantitative results for MDA content and SOD activity in the whole brain. (C) Bands of SOD-1 protein detected by western blot assay. (D) Quantitative results for SOD-1 protein expression, which was normalized to β-actin. Data are represented as the mean ± SD (n= 6 in each group). *P< 0.05 (Student's t-test). miR-29b: MicroRNA-29b; MCAO: middle cerebral artery occlusion; MDA: malondialdehyde; SOD: superoxide dismutase.

Figure 7

MiR-29b regulates the PI3K/Akt/Bax signaling pathway in MCAO model mice. (A) Bands of protein in the PI3K/Akt/Bax signaling pathway detected by western blot assay. (B) Quantitative results of protein levels for p-PI3K/PI3K (B), p-Akt/Akt (C), and Bcl-2/Bax (D) in the whole brain. Data are presented as the mean ± SD (n= 6 in each group). *P< 0.05 (Student's t-test). MCAO: Middle cerebral artery occlusion; miR-29b: microRNA-29b; PI3K: phosphoinositide 3-kinase.