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. 2017 Feb 15;37(7):1797-1806.
doi: 10.1523/JNEUROSCI.3389-16.2017. Epub 2017 Jan 16.

Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

Xuejing Zhang  1 , Xuelian Tang  1 , Kai Liu  1 , Milton H Hamblin  2 , Ke-Jie Yin  3
Affiliations

Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

Xuejing Zhang et al. J Neurosci. .

Abstract

The study was designed to determine the role of long noncoding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (Malat1), in ischemic stroke outcome. Primary mouse brain microvascular endothelial cells (BMECs) were cultured and treated with Malat1 GapmeR before 16 h oxygen and glucose depravation (OGD). Cell death was assayed by LDH and MTT methods. Malat1 knock-out and wild-type mice were subjected to 1 h of middle cerebral artery occlusion (MCAO) and 24-72 h of reperfusion. To explore the underlying mechanism, apoptotic and inflammatory factors were measured by qPCR, ELISA, and Western blotting. The physical interaction between Malat1 and apoptotic or inflammatory factors was measured by RNA immunoprecipitation. Increased Malat1 levels were found in cultured mouse BMECs after OGD as well as in isolated cerebral microvessels in mice after MCAO. Silencing of Malat1 by Malat1 GapmeR significantly increased OGD-induced cell death and Caspase 3 activity in BMECs. Silencing of Malat1 also significantly aggravated OGD-induced expression of the proapoptotic factor Bim and proinflammatory cytokines MCP-1, IL-6, and E-selectin. Moreover, Malat1 KO mice presented larger brain infarct size, worsened neurological scores, and reduced sensorimotor functions. Consistent with in vitro findings, significantly increased expression of proapoptotic and proinflammatory factors was also found in the cerebral cortex of Malat1 KO mice after ischemic stroke compared with WT controls. Finally, we demonstrated that Malat1 binds to Bim and E-selectin both in vitro and in vivo Our study suggests that Malat1 plays critical protective roles in ischemic stroke.SIGNIFICANCE STATEMENT Accumulative studies have demonstrated the important regulatory roles of microRNAs in vascular and neural damage after ischemic stroke. However, the functional significance and mechanisms of other classes of noncoding RNAs in cerebrovascular pathophysiology after stroke are less studied. Here we demonstrate a novel role of Malat1, a long noncoding RNA that has been originally identified as a prognostic marker for non-small cell lung cancer, in cerebrovascular pathogenesis of ischemic stroke. Our experiments have provided the first evidence that Malat1 plays anti-apoptotic and anti-inflammatory roles in brain microvasculature to reduce ischemic cerebral vascular and parenchymal damages. Our studies also suggest that lncRNAs can be therapeutically targeted to minimize poststroke brain damage.

Keywords: Malat1; apoptosis; cerebral ischemia; cerebral vascular endothelial cell; inflammation; stroke.

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Figures

Figure 1.
Figure 1.
Malat1 expression in mouse BMEC cultures and mouse cerebral microvessels following focal cerebral ischemia. A, RNA-seq and qPCR data showing the expression of Malat1 in cultured BMECs. Malat1 was significantly induced after OGD treatment. B, Malat1 level was also examined in isolated cerebral microvessels (Cereb. Ves.) and cerebral cortex (Cereb. Ctx.) in mice subjected to 1 h MCAO and 24 h reperfusion (n = 3). Significantly increased expression of Malat1 was found in isolated cerebral microvessels after MCAO. C, In situ hybridization detection of Malat1 nuclear distribution (arrows) in BMECs. Data are mean ± SEM. *p < 0.05 versus Non-OGD or Sham group. Scale bar, 20 μm.
Figure 2.
Figure 2.
Silencing of Malat1 by LNA-GapmeRs increases OGD-induced mouse BMEC cell death. GapmeRs are DNA oligonucleotides with LNA residues at the 3′ and 5′ ends that induce RNase H-mediated degradation of nuclear RNA. GapmeRs targeting Malat1 or scrambled control GapmeRs were transfected at 50 nm. A, Images were taken 48 h after transfection. GFP+ cells indicated successfully transfected cells. B, qPCR showed significantly reduced Malat1 levels in BMECs after transfection in the absence or presence of 16 h OGD exposure. C, D, Exposure to OGD results in obvious BMEC cell death detected by LDH assay (C) and MTT assay (D). Increased OGD-induced cell death in Malat1 GapmeR-transfected BMECs compared with Malat1 Ctrl-transfected cells. E, Exposure to OGD increases caspase 3 activation in BMEC cells transfected with Malat1 GapmeR. F, qPCR showed significantly reduced Malat1 levels in N2A cells after transfection in the absence or presence of 4 h OGD exposure. G, H, Exposure to OGD resulted in remarkable N2A cell death detected by LDH (G) and MTT assay (H). However, silencing of Malat1 by GapmeR had no effects on OGD-induced N2A cell death. Data are mean ± SEM (n = 3). *p < 0.05 versus Malat1 control + Non-OGD or OGD group. Scale bar, 50 μm.
Figure 3.
Figure 3.
Effects of Malat1 genetic deficiency on ischemic infarction and neurological outcomes. Malat1 KO and littermate control (Malat1 WT) mice were subjected to 1 h MCAO and 24 h reperfusion. A, The 2% TTC-stained coronal sections are shown at different brain levels posterior to the frontal pole. B, C, Quantitative analysis was performed on infarct volume (B) and neurological deficits (C) in mice after stroke. In comparison with Malat1 WT mice, Malat1 KO mice showed larger ischemia-induced brain infarction (n = 11) and worsened neurological outcomes (n = 11). Data are mean ± SD. *p < 0.05 versus the WT group.
Figure 4.
Figure 4.
Genetic deletion of Malat1 gene aggravates sensorimotor functions of mice after MCAO. A–D, Focal cerebral ischemia was induced in Malat1 WT and Malat1 KO mice by 1 h MCAO followed by 1–7 d reperfusion. Ischemic mice (n = 8) were subjected to adhesive tape removal (A, B), foot fault (C), and cylinder (D) tests for examination of sensorimotor functions. In comparison with Malat1 WT mice, Malat1 KO mice showed increased touch time, removal time, fault steps, and less balanced use of both limbs after MCAO. Data are mean ± SD. *p < 0.05 versus the Malat1 WT group.
Figure 5.
Figure 5.
The effect of Malat1 silencing on proapoptotic factors in mouse BMECs after OGD and in mouse brain after focal cerebral ischemia. A, Cultured BMECs were transfected by LNA-Malat1 GapmeR and scramble control for 48 h, and then subjected to 16 h OGD. The expression levels of Bim and Bax were determined by qPCR and normalized to cyclophilin (n = 3). B, The protein levels of Bim and Bax were determined by Western blotting, with β-actin as the loading control. C, Stroke was induced in Malat1 WT and Malat1 KO mice by 1 h MCAO followed by 24 h reperfusion. Expression levels of Bim and Bax were determined by qPCR and normalized to cyclophilin (n = 6/group). D, The protein levels of Bim and Bax were determined, with β-actin as the loading control. Silencing of Malat1 increased the expression of the proapoptotic factor Bim in cultured BMECs and in the mouse ischemic brain regions after MCAO. Experiments were repeated three times, and representative blots are displayed. Data are mean ± SEM. *p < 0.05 versus Malat1 Ctrl + ODG or Malat1 WT + MCAO group. #p < 0.05 versus Malat1 Ctrl + Non-OGD or Malat1 WT + sham group.
Figure 6.
Figure 6.
Effects of Malat1 silencing on major proinflammatory cytokines in mouse BMECs after OGD. A–C, Cultured BMECs were transfected with LNA-Malat1 GapmeR and scramble control for 48 h, and then subjected to 16 h OGD. The expression levels of E-selectin (A), MCP-1 (B), and IL-6 (C) were determined by qPCR and normalized to cyclophilin (n = 3). D, The protein level of E-selectin was determined by Western blotting, with β-actin as the loading control. Experiments were repeated three times, and representative blots are displayed. E, F, Protein levels of MCP-1 (E) and IL-6 (F) in Malat1 Ctrl and Malat1 GapmeR-treated BMEC culture medium were analyzed by ELISA (n = 3). Silencing of Malat1 by GapmeR treatment increased the expression of proinflammatory cytokines MCP-1, IL-6, and E-selectin in cultured BMECs after 16 h OGD. Data are mean ± SD. *p < 0.05 versus Malat1 Ctrl + OGD group. #p < 0.05 versus Malat1 Ctrl + Non-OGD group.
Figure 7.
Figure 7.
Effects of Malat1 genetic deficiency on proinflammatory cytokines in mouse brain after focal cerebral ischemia. A–C, Stroke was induced in Malat1 WT and KO mice by 1 h MCAO followed by 24 h reperfusion. Total RNA was extracted from Malat1 WT and Malat1 KO mouse cortex 24 h after MCAO, and expression levels of E-selectin (A), MCP-1 (B), and IL-6 (C) were determined by qPCR and normalized to cyclophilin (n = 6/group). D, Total protein was extracted and subjected to gel electrophoresis. The protein levels of E-selectin, MCP-1, and IL-6 were determined, with β-actin as the loading control. Increased expression levels of the proinflammatory cytokines MCP-1, IL-6, and E-selectin were induced in Malat1 KO mice 24 h after MCAO compared with Malat1 WT mice. Experiments were repeated three times, and representative blots are displayed. Data are mean ± SEM. *p < 0.05 versus Malat1 WT + MCAO group. #p < 0.05 versus Malat1 WT + Sham group.
Figure 8.
Figure 8.
Malat1 physically associates with Bim and E-selectin in mouse BMECs and mouse brains. A, B, Interaction between Malat1 and Bim or E-selectin revealed by RIP experiments. Total extracts of cultured BMECs after 16 h OGD (A) and mouse brains after 24 h MCAO (B) were immunoprecipitated with control IgG, anti-Bim, or anti-E-selectin antibody, and the complexes were analyzed for the presence of Malat1 by qPCR. Fold enrichment over the normal rabbit IgG was calculated by the ΔΔCt method (n = 3). Data are mean ± SEM of two independent experiments with triplicate wells. *p < 0.05 versus IgG group.
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