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Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction
Published on: May 7, 2015
Circ_0001360 absence alleviates oxygen-glucose deprivation/reoxygenation-induced SK-N-SH cell injury via controlling
Fang Lu1, Linhong Mo1, Aixian Liu1
1Department of Neurology, Beijing Rehabilitation Hospital, Capital Medical University, Beijing, China.
Insights
Circular RNA circ_0001360 exacerbates ischemic stroke injury by targeting miR-671-5p and activating the NF-κB pathway. Its inhibition protects against oxygen-glucose deprivation/reoxygenation-induced cell damage.
Area of Science:
- Molecular Biology
- Genetics
- Neuroscience
Background:
- Circular RNAs (circRNAs) play regulatory roles in human diseases.
- Ischemic stroke (IS) involves complex molecular mechanisms.
- Limited research exists on circRNA involvement in IS.
Purpose of the Study:
- To investigate the role of circ_0001360 in in vitro cell models of ischemic stroke.
- To elucidate the molecular mechanisms underlying circ_0001360's function in IS pathology.
Main Methods:
- Simulated IS conditions using oxygen-glucose deprivation/reoxygenation (OGD/R) on SK-N-SH cells.
- Quantitative real-time PCR (qPCR) and western blot for gene and protein expression analysis.
- Assays for cell viability (CCK-8), proliferation (EdU), apoptosis (flow cytometry), and pathway activity (NF-κB).
- Dual-luciferase reporter and RIP assays to confirm molecular interactions.
Main Results:
- Circ_0001360 expression was upregulated in OGD/R-treated SK-N-SH cells.
- Circ_0001360 knockdown alleviated OGD/R-induced cell impairment, apoptosis, and inflammation.
- Circ_0001360 targeted miR-671-5p, which in turn regulated BMF expression.
- Circ_0001360 knockdown inhibited the NF-κB pathway, an effect reversed by miR-671-5p inhibition or BMF overexpression.
Conclusions:
- Circ_0001360 promotes OGD/R-induced cell injury in IS models.
- The mechanism involves targeting the miR-671-5p/BMF axis and activating the NF-κB pathway.
- Circ_0001360 is a potential therapeutic target for ischemic stroke.
Background:
The regulatory potency of circular RNA (circRNA) has been acknowledged in multiple human diseases, including ischaemic stroke (IS). However, only a few circRNAs were investigated in this disorder. We aimed to uncover the role of circ_0001360 in cell models of IS in vitro.
Methods:
SK-N-SH cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate IS pathology conditions in vitro. Quantitative real-time PCR (qPCR) and western blot were applied for expression detection. Cell viability, proliferation and apoptosis were investigated by CCK-8, EdU and flow cytometry assays. The predicted binding of miR-671-5p to circ_0001360 or BMF 3'UTR was validated by dual-luciferase reporter and RIP assays. Proteins on the NF-κB pathway were quantified by western blot to assess NF-κB pathway activity.
Results:
Circ_0001360 was upregulated in SK-N-SH cells after OGD/R treatment. OGD/R provoked SK-N-SH cell growth impairment, apoptosis and inflammation, while circ_0001360 knockdown relieved these injuries. Circ_0001360 targeted miR-671-5p, and miR-671-5p deficiency recovered SK-N-SH cell injury that was repressed by circ_0001360 knockdown. MiR-671-5p directly combined with BMF and repressed BMF expression. Accordingly, circ_0001360 targeted miR-671-5p to regulate the expression of BMF. Circ_0001360 knockdown weakened the phosphorylated levels of P65 and IκBα, while further miR-671-5p deficiency or BMF overexpression restored their expression levels.
Conclusion:
Circ_0001360 contributed to OGD/R-caused SK-N-SH cell injury via targeting the miR-671-5p/BMF network and activating the NF-κB pathway, thus participating in the development of IS.
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