Journal: The Journal of Neuroscience

Article Title: Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

doi: 10.1523/JNEUROSCI.3389-16.2017

Figure Lengend Snippet: 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.

Article Snippet: The expression and nuclear localization of Malat1 were also shown in BMECs by in situ hybridization using Malat1 FITC-labeled LNA probes (Exiqon) but not by the control probe ( C ).

Techniques: Expressing, RNA Sequencing Assay, Cell Culture, Isolation, In Situ Hybridization

Journal: The Journal of Neuroscience

Article Title: Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

doi: 10.1523/JNEUROSCI.3389-16.2017

Figure Lengend Snippet: 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.

Article Snippet: The expression and nuclear localization of Malat1 were also shown in BMECs by in situ hybridization using Malat1 FITC-labeled LNA probes (Exiqon) but not by the control probe ( C ).

Techniques: Transfection, Lactate Dehydrogenase Assay, MTT Assay, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

doi: 10.1523/JNEUROSCI.3389-16.2017

Figure Lengend Snippet: 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.

Article Snippet: The expression and nuclear localization of Malat1 were also shown in BMECs by in situ hybridization using Malat1 FITC-labeled LNA probes (Exiqon) but not by the control probe ( C ).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

doi: 10.1523/JNEUROSCI.3389-16.2017

Figure Lengend Snippet: 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.

Article Snippet: The expression and nuclear localization of Malat1 were also shown in BMECs by in situ hybridization using Malat1 FITC-labeled LNA probes (Exiqon) but not by the control probe ( C ).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

doi: 10.1523/JNEUROSCI.3389-16.2017

Figure Lengend Snippet: 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.

Article Snippet: The expression and nuclear localization of Malat1 were also shown in BMECs by in situ hybridization using Malat1 FITC-labeled LNA probes (Exiqon) but not by the control probe ( C ).

Techniques: Cell Culture, Transfection, Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

doi: 10.1523/JNEUROSCI.3389-16.2017

Figure Lengend Snippet: 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.

Article Snippet: The expression and nuclear localization of Malat1 were also shown in BMECs by in situ hybridization using Malat1 FITC-labeled LNA probes (Exiqon) but not by the control probe ( C ).

Techniques: Cell Culture, Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Neuroscience

Article Title: Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

doi: 10.1523/JNEUROSCI.3389-16.2017

Figure Lengend Snippet: 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.

Article Snippet: The expression and nuclear localization of Malat1 were also shown in BMECs by in situ hybridization using Malat1 FITC-labeled LNA probes (Exiqon) but not by the control probe ( C ).

Techniques: Expressing, Nucleic Acid Electrophoresis

Journal: The Journal of Neuroscience

Article Title: Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke

doi: 10.1523/JNEUROSCI.3389-16.2017

Figure Lengend Snippet: 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.

Article Snippet: The expression and nuclear localization of Malat1 were also shown in BMECs by in situ hybridization using Malat1 FITC-labeled LNA probes (Exiqon) but not by the control probe ( C ).

Techniques: Cell Culture, Immunoprecipitation