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. 2016 Mar:277:162-170.
doi: 10.1016/j.expneurol.2015.12.014. Epub 2015 Dec 31.

"V体育2025版" Altered long non-coding RNA transcriptomic profiles in brain microvascular endothelium after cerebral ischemia

J Zhang  1 , L Yuan  1 , X Zhang  2 , M H Hamblin  3 , T Zhu  1 , F Meng  4 , Y Li  5 , Y E Chen  6 , K J Yin  7
Affiliations

Altered long non-coding RNA transcriptomic profiles in brain microvascular endothelium after cerebral ischemia

J Zhang et al. Exp Neurol. 2016 Mar.

Abstract

The brain endothelium is an important therapeutic target for the inhibition of cerebrovascular dysfunction in ischemic stroke. Previously, we documented the important regulatory roles of microRNAs in the cerebral vasculature, in particular the cerebral vascular endothelium. However, the functional significance and molecular mechanisms of other classes of non-coding RNAs in the regulation of cerebrovascular endothelial pathophysiology after stroke are completely unknown. Using RNA sequencing (RNA-seq) technology, we profiled long non-coding RNA (lncRNA) expressional signatures in primary brain microvascular endothelial cells (BMECs) after oxygen-glucose deprivation (OGD), an in vitro mimic of ischemic stroke conditions. After 16h of OGD exposure, the expression levels for 362 of the 10,677 lncRNAs analyzed changed significantly, including a total of 147 lncRNAs increased and 70 lncRNAs decreased by more than 2-fold. Among them, the most highly upregulated lncRNAs include Snhg12, Malat1, and lnc-OGD 1006, whereas the most highly downregulated lncRNAs include 281008D09Rik, Peg13, and lnc-OGD 3916. Alteration of the most highly upregulated/downregulated ODG-responsive lncRNAs was further confirmed in cultured BMECs after OGD as well as isolated cerebral microvessels in mice following transient middle cerebral artery occlusion (MCAO) and 24h reperfusion by the quantitative real-time PCR approach. Moreover, promoter analysis of altered ODG-responsive endothelial lncRNA genes by bioinformatics showed substantial transcription factor binding sites on lncRNAs, implying potential transcriptional regulation of those lncRNAs. These findings are the first to identify OGD-responsive brain endothelial lncRNAs, which suggest potential pathological roles for these lncRNAs in mediating endothelial responses to ischemic stimuli. Endothelial-selective lncRNAs may function as a class of novel master regulators in cerebrovascular endothelial pathologies after ischemic stroke VSports手机版. .

Keywords: Brain microvascular endothelial cells; Ischemic stroke; Long non-coding RNAs; Oxygen–glucose deprivation; RNA sequencing; Transcriptome V体育安卓版. .

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Figures

Figure 1
Figure 1
Brain microvascular endothelial cell cultures. Phase-contrast light microscopy shows primary mouse BMEC cultures. Immunofluorescent stain using antibody against brain vascular endothelial cell marker, CD31 and BBB markers, Claudin 5 and Occludin reveals typical circumcellular signals of CD31, Claudin 5, and Occludin. Scale bars are indicated.
Figure 2
Figure 2
Schematic representation of RNA-seq experiments and bioinformatics analysis.
Figure 3
Figure 3
Altered lncRNA and mRNA profiles in mouse BMECs after OGD. Heatmap and hierarchical clustering analysis of 10,677 lncRNAs (A) and 43,705 protein-coding RNAs (B) that were differentially expressed (P<0.045 and FDR<0.05) in mouse BMECs 16 hours after OGD exposure. Four independent biological replicates were analyzed for non-OGD and OGD conditions. The clustering tree for lncRNAs and protein-coding RNAs is shown at the top. The expression values are represented in shades of red and green, indicating expression above and below the median value across all samples (log scale 2, from −2.00 to +2.00), respectively.
Figure 4
Figure 4
PCR verification of expression profiles of lncRNA in mouse BMECs after OGD. The relative expression levels of most highly changed lncRNAs as indicated were examined using qRT-PCR in mouse BMECs that were treated with OGD for 16 hours. Data from RNA-seq were well-matched to quantitative PCR results. Data are expressed as mean ± SEM from 3 separate experiments.
Figure 5
Figure 5
Altered lncRNA expression profiles in cerebral microvasculature and brain in mice after focal cerebral ischemia. (A) Brain microvessels were isolated from mouse cerebral cortex and the expression of multiple neurovascular cell markers was analyzed by qPCR. Characterized high expression of brain EC markers, ZO-1, claudin-5, CD31, and vWF with much lower levels of the astrocyte marker, GFAP and pericyte marker, platelet-derived growth factor receptor-β, indicates a relative high EC abundance. (B) QPCR data demonstrated that three most highly upregulated and downregulated OGD-responsive lncRNAs as indicated are also significantly increased and reduced respectively in cerebral microvessels (Cereb Ves) but not in brain parenchyma (cerebral cortex, Ctx) in mice after 1h MCAO followed by 24h reperfusion. Data are expressed as mean + SEM. *p<0.05 vs. Sham or ZO-1 group.
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