Background/aims: With advances in RNA-sequencing (RNA-seq), exploring the expression and transcripts of different classes of genes are now possible. Our purpose was to analyse to the long non-coding RNAs (lncRNAs) in mouse brain microvascular endothelial cells (bEnd.3) after oxygen-glucose deprivation/re-oxygenation (OGD/R).
Methods: RNA-seq was employed to explore the expression of lncRNAs, and quantitative real-time PCR (qRT-PCR) was used to identify the results of RNA-seq. Furthermore, the biological functions of the lncRNAs were identified by cell viability, wound healing, transwell, tube formation and immunofluorescent staining assays. Finally, the molecular mechanisms involving the differentially expressed lncRNAs were further explored by bioinformatics and Western blotting (WB).
Results: In total, 2710 lncRNAs were found, 33 of which were significantly differentially expressed, with 18 upregulated lncRNAs and 15 downregulated lncRNAs in brain microvascular endothelial cells following OGD/R. Among the dysregulated genes, G protein-coupled receptor 137b-pseudogene (Gpr137b-ps), predicted gene 32856 (Gm32856), small nucleolar RNA host gene 17 (snhg17), chaperonin containing Tcp1 and subunit 6a (Cct6a) were significantly upregulated lncRNAs; this finding was further validated using qRT-PCR. Moreover, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes (KEGG) pathway analyses were employed to decipher the potential target genes and signaling pathways of the differentially expressed lncRNAs. Finally, we selected pseudogene-expressed lncRNA Gpr137b-ps as a candidate gene, and report for the first time that pseudogenes can mediate angiogenesis and their potential target genes, namely, 15-lipoxygenase1 (15-LOX1), Signal Transducer and Activator of Transcription 3 (S TAT3) and vascular endothelial growth factor (VEGF).
Conclusion: Therefore, our study revealed that Gpr137b-ps plays critical roles in the process of angiogenesis, suggesting avenues for the development of future therapeutic strategies that contribute to promoting angiogenesis following I/R.
Keywords: Angiogenesis; Endothelial cells; Long non-coding RNAs; Oxygen-glucose deprivation/reoxygenation; Pseudogenes; RNA-sequencing.
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