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Updated: Jul 14, 2026
Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model
Published on: August 29, 2012
Circvrk1 downregulation attenuates brain microvascular endothelial cell damage induced by oxygen-glucose deprivation
Lina Tan1, Lingjun Wang2, Jiajun Liu3
1Department of Neurology, The First Affiliated Hospital of Qiqihar Medical University, Hengtai B District Fularji District, Qiqihar, 161000, Heilongjiang, China. Tanlina79140405@126.com.
Insights
Circular RNA VRK1 (circVRK1) exacerbates ischemic stroke (IS) by regulating the miR-150-5p/MLLT1 pathway. Reducing circVRK1 protects brain microvascular endothelial cells from injury, offering potential therapeutic targets for IS.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Circular RNAs (circRNAs) are increasingly recognized for their regulatory roles in various biological processes.
- Ischemic stroke (IS) remains a leading cause of disability, necessitating the identification of novel therapeutic targets.
- Understanding the molecular mechanisms underlying IS pathogenesis is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the role and mechanism of circVRK1 in oxygen-glucose deprivation (OGD)-induced human brain microvascular endothelial cell (HBMEC) injury, a model for IS.
- To elucidate the interaction between circVRK1, miR-150-5p, and MLLT1 in the context of IS.
- To explore circVRK1 as a potential therapeutic target for IS.
Main Methods:
- Establishment of an in vitro IS model using OGD-challenged HBMECs.
- Quantitative real-time PCR to measure circRNA, mRNA, and microRNA expression levels.
- Cellular assays including CCK-8, Transwell, wound-healing, and tube formation assays to assess cell viability, migration, and angiogenesis.
- Western blot analysis for protein expression, ELISA for oxidative stress, and flow cytometry for cell death.
- Dual-luciferase reporter, RIP, and RNA pull-down assays to confirm molecular interactions.
Main Results:
- CircVRK1 expression was significantly upregulated in OGD-treated HBMECs.
- Knockdown of circVRK1 attenuated OGD-induced HBMEC injury, improving cell viability, migration, and angiogenesis while reducing cell death, inflammation, and oxidative stress.
- CircVRK1 acted as a sponge for miR-150-5p, and miR-150-5p silencing reversed the protective effects of circVRK1 deficiency.
- MLLT1 was identified as a target of miR-150-5p, and its overexpression counteracted the protective effects of miR-150-5p.
- CircVRK1 knockdown protected HBMECs from OGD-induced injury by modulating the miR-150-5p/MLLT1 pathway.
Conclusions:
- CircVRK1 plays a critical role in exacerbating HBMEC injury during ischemic stroke.
- The circVRK1/miR-150-5p/MLLT1 axis is a key molecular mechanism involved in IS pathogenesis.
- CircVRK1 represents a promising therapeutic target for ischemic stroke treatment.
Abstract:
The pivotal regulatory role of circular RNAs (circRNAs) in ischemic stroke (IS) has been expounded. The study aimed to probe the exact role and underlying mechanism of circVRK1 in oxygen-glucose deprivation (OGD)-induced human brain microvascular endothelial cells (HBMECs) injury. HBMECs challenged by OGD were used as in vitro models of IS. Quantitative real-time PCR was used to examine the levels of circVRK1, vaccinia-related kinase 1 (VRK1), miR-150-5p and MLLT1 mRNA. Cell viability, migration angiogenesis ability and death were evaluated by Cell counting kit-8 assay, transwell assay, wound-healing assay, tube formation assay and flow cytometry analysis. All the protein levels were monitored by western blot assay. Enzyme-linked immunosorbent assay was conducted for examining cell oxidative stress. Dual-luciferase reporter assay, RIP assay and RNA pull-down assay were performed to verify the combination between miR-150-5p and circVRK1 or MLLT1. CircVRK1 was upregulated in OGD-treated HBMECs. CircVRK1 knockdown alleviated OGD-caused effects on HBMECs migration, angiogenesis, death, inflammatory response and oxidative stress. Furthermore, circVRK1 could sponge miR-150-5p, and miR-150-5p silencing also mitigated the impact of circVRK1 deficiency on OGD-evoked injury. Besides, MLLT1 acted as a molecular target of miR-150-5p, and the protective influence of miR-150-5p on OGD-induced cell damage was overturned by MLLT1 introduction. CircVRK1 knockdown weakened OGD-evoked injury in HBMECs through modulating miR-150-5p/MLLT1 pathway, and this might supply new insights and probable targets for IS treatment.
