Zhi-Wen Zhou1, Li-Jun Zheng2, Xiang Ren1, Ai-Ping Li1, Wen-Sheng Zhou3. 1. The Third Department of Neurology, Hunan Provincial People's Hospital(The First Affiliated Hospital of Hunan Normal University), Changsha 410001, PR China. 2. Department of Rehabilitation, Hunan Provincial People's Hospital(The First Affiliated Hospital of Hunan Normal University), Changsha 410001, PR China. 3. The Third Department of Neurology, Hunan Provincial People's Hospital(The First Affiliated Hospital of Hunan Normal University), Changsha 410001, PR China. Electronic address: zhouwensheng09@163.com.
Abstract
OBJECTIVE: The present study was designed to investigate the mechanism by which lncRNA NEAT1 regulates survival and angiogenesis in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). METHODS: OGD-treated BMECs were used to mimic cerebral ischaemia in vitro. The expression of lncRNA NEAT1 and miR-377 and proteins including VEGFA, SIRT1, and BCL-XL were measured by real-time quantitative PCR (qRT-PCR) and western blot, respectively. Cell viability and caspase 3 activity of BMECs under different conditions were determined using MTT and caspase activity assays, respectively. Matrigel-based angiogenesis assays were employed to evaluate the effect of lncRNA NEAT1 on angiogenesis. A dual-luciferase reporter assay was used to validate direct binding of miR-377 to putative targets. RESULTS: OGD exposure reduced the cell viability of BMECs. Upregulation of lncRNA NEAT1 and downregulation of miR-377 were also observed under OGD conditions. Knockdown of lncRNA NEAT1 inhibited angiogenesis and aggravated apoptosis in OGD-induced BMECs. Meanwhile, the expression level of miR-377 was upregulated while its downstream targets (VEGFA, SIRT1, and BCL-XL) were downregulated after lncRNA NEAT1 knockdown. Furthermore, miR-377 inhibited the angiogenesis and survival of OGD-induced BMECs. The expression of VEGFA, SIRT1, and BCL-XL were all attenuated by miR-377 overexpression. The dual-luciferase reporter assay proved miR-377 targeted the 3' UTR sequences of lncRNA NEAT1, VEGFA, SIRT1, and BCL-XL. CONCLUSION: lncRNA NEAT1 facilitated the survival and angiogenesis of OGD-induced BMECs via targeting miR-377 and promoting the expression of VEGFA, SIRT1, and BCL-XL, suggesting that lncRNA NEAT1 could be a promising target for cerebral ischaemia treatment.
OBJECTIVE: The present study was designed to investigate the mechanism by which lncRNA NEAT1 regulates survival and angiogenesis in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). METHODS: OGD-treated BMECs were used to mimic cerebral ischaemia in vitro. The expression of lncRNA NEAT1 and miR-377 and proteins including VEGFA, SIRT1, and BCL-XL were measured by real-time quantitative PCR (qRT-PCR) and western blot, respectively. Cell viability and caspase 3 activity of BMECs under different conditions were determined using MTT and caspase activity assays, respectively. Matrigel-based angiogenesis assays were employed to evaluate the effect of lncRNA NEAT1 on angiogenesis. A dual-luciferase reporter assay was used to validate direct binding of miR-377 to putative targets. RESULTS: OGD exposure reduced the cell viability of BMECs. Upregulation of lncRNA NEAT1 and downregulation of miR-377 were also observed under OGD conditions. Knockdown of lncRNA NEAT1 inhibited angiogenesis and aggravated apoptosis in OGD-induced BMECs. Meanwhile, the expression level of miR-377 was upregulated while its downstream targets (VEGFA, SIRT1, and BCL-XL) were downregulated after lncRNA NEAT1 knockdown. Furthermore, miR-377 inhibited the angiogenesis and survival of OGD-induced BMECs. The expression of VEGFA, SIRT1, and BCL-XL were all attenuated by miR-377 overexpression. The dual-luciferase reporter assay proved miR-377 targeted the 3' UTR sequences of lncRNA NEAT1, VEGFA, SIRT1, and BCL-XL. CONCLUSION: lncRNA NEAT1 facilitated the survival and angiogenesis of OGD-induced BMECs via targeting miR-377 and promoting the expression of VEGFA, SIRT1, and BCL-XL, suggesting that lncRNA NEAT1 could be a promising target for cerebral ischaemia treatment.