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Abstract <p>Background: Cerebral vasospasm (CVS) following aneurysmal subarachnoid hemorrhage (aSAH) is a major cause of disability and mortality, yet its pathogenesis remains incompletely understood. long non-coding RNA HOX Transcript Antisense RNA (lnc-HOTAIR) is involved in the regulation of endothelial cell function, but its role and molecular mechanism in post-aSAH CVS have not been elucidated. This study aimed to investigate whether lnc-HOTAIR participates in the development of CVS after aSAH by modulating Methylenetetrahydrofolate Reductase (MTHFR) and the Notch signaling pathway, thereby affecting vascular endothelial cells (VECs) injury. Methods: RT-qPCR was used to detect the expression levels of lnc-HOTAIR and MTHFR in the basilar artery of aSAH and CVS rats. Lentivirus-mediated shRNA knockdown or pcDNA overexpression was employed to manipulate lnc-HOTAIR and MTHFR expression both in vivo and in vitro. CCK-8 assay, flow cytometry, Western blotting, and H&amp;E staining were used to assess VEC proliferation, apoptosis, protein expression, and basilar artery morphological changes. Dual-luciferase reporter assay, RIP, and RNA pull-down assays were performed to verify the direct binding between lnc-HOTAIR and MTHFR. Jagged1/Fc (a Notch pathway activator) and DAPT (a Notch pathway inhibitor) were used to intervene in Notch signaling, and their effects on VEC proliferation and apoptosis were examined. Homocysteine (Hcy) was used to simulate VEC injury in vitro. Results: lnc-HOTAIR expression was significantly upregulated in the basilar artery of aSAH and CVS rats and was negatively correlated with MTHFR expression. In vivo experiments showed that lnc-HOTAIR overexpression exacerbated bleeding, basilar artery luminal narrowing, and vessel wall thickening in aSAH rats, whereas lnc-HOTAIR silencing alleviated these changes. In vitro experiments revealed that Hcy upregulated lnc-HOTAIR expression and downregulated MTHFR expression in VECs in a concentration-dependent manner. Mechanistic studies demonstrated that lnc-HOTAIR directly bound to MTHFR and inhibited its expression. Knockdown of lnc-HOTAIR or overexpression of MTHFR reversed Hcy-induced VEC proliferation inhibition and apoptosis, whereas lnc-HOTAIR overexpression attenuated the protective effect of MTHFR. Furthermore, knockdown of lnc-HOTAIR or overexpression of MTHFR activated the Notch signaling pathway and upregulated VEGF expression, thereby inhibiting Hcy-induced VEC apoptosis and promoting proliferation. Conclusion: lnc-HOTAIR is upregulated in post-aSAH CVS and directly binds to and inhibits MTHFR expression, leading to suppression of the Notch signaling pathway and VEGF, which ultimately promotes VEC apoptosis and inhibits proliferation, thereby exacerbating cerebral vasospasm. Knockdown of lnc-HOTAIR reverses these effects and exerts a protective role.</p> Graphical Abstract <p></p>

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lnc-HOTAIR inhibits MTHFR expression to regulate endothelial cell proliferation and apoptosis in aSAH-induced cerebral vasospasm

  • 期刊论文

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  • 发表日期：

    2026年8月1日

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  • Pingbo Wei,
  • Dihong Chen,
  • Yangyun Han,
  • Qin Du,
  • Le Luo,
  • Chaohua Wang,
  • Jiaxin Liu

摘要

Abstract

Background: Cerebral vasospasm (CVS) following aneurysmal subarachnoid hemorrhage (aSAH) is a major cause of disability and mortality, yet its pathogenesis remains incompletely understood. long non-coding RNA HOX Transcript Antisense RNA (lnc-HOTAIR) is involved in the regulation of endothelial cell function, but its role and molecular mechanism in post-aSAH CVS have not been elucidated. This study aimed to investigate whether lnc-HOTAIR participates in the development of CVS after aSAH by modulating Methylenetetrahydrofolate Reductase (MTHFR) and the Notch signaling pathway, thereby affecting vascular endothelial cells (VECs) injury. Methods: RT-qPCR was used to detect the expression levels of lnc-HOTAIR and MTHFR in the basilar artery of aSAH and CVS rats. Lentivirus-mediated shRNA knockdown or pcDNA overexpression was employed to manipulate lnc-HOTAIR and MTHFR expression both in vivo and in vitro. CCK-8 assay, flow cytometry, Western blotting, and H&E staining were used to assess VEC proliferation, apoptosis, protein expression, and basilar artery morphological changes. Dual-luciferase reporter assay, RIP, and RNA pull-down assays were performed to verify the direct binding between lnc-HOTAIR and MTHFR. Jagged1/Fc (a Notch pathway activator) and DAPT (a Notch pathway inhibitor) were used to intervene in Notch signaling, and their effects on VEC proliferation and apoptosis were examined. Homocysteine (Hcy) was used to simulate VEC injury in vitro. Results: lnc-HOTAIR expression was significantly upregulated in the basilar artery of aSAH and CVS rats and was negatively correlated with MTHFR expression. In vivo experiments showed that lnc-HOTAIR overexpression exacerbated bleeding, basilar artery luminal narrowing, and vessel wall thickening in aSAH rats, whereas lnc-HOTAIR silencing alleviated these changes. In vitro experiments revealed that Hcy upregulated lnc-HOTAIR expression and downregulated MTHFR expression in VECs in a concentration-dependent manner. Mechanistic studies demonstrated that lnc-HOTAIR directly bound to MTHFR and inhibited its expression. Knockdown of lnc-HOTAIR or overexpression of MTHFR reversed Hcy-induced VEC proliferation inhibition and apoptosis, whereas lnc-HOTAIR overexpression attenuated the protective effect of MTHFR. Furthermore, knockdown of lnc-HOTAIR or overexpression of MTHFR activated the Notch signaling pathway and upregulated VEGF expression, thereby inhibiting Hcy-induced VEC apoptosis and promoting proliferation. Conclusion: lnc-HOTAIR is upregulated in post-aSAH CVS and directly binds to and inhibits MTHFR expression, leading to suppression of the Notch signaling pathway and VEGF, which ultimately promotes VEC apoptosis and inhibits proliferation, thereby exacerbating cerebral vasospasm. Knockdown of lnc-HOTAIR reverses these effects and exerts a protective role.

Graphical Abstract