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Updated: Mar 9, 2026
Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke
Published on: January 3, 2025
Influence of miR-155 on Cell Apoptosis in Rats with Ischemic Stroke: Role of the Ras Homolog Enriched in Brain
Guoping Xing1,2, Zengxiang Luo3, Chi Zhong2
1Department of Neurology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China (mainland).
Abstract:
BACKGROUND We designed and carried out this study to examine the role of miR-155 and the Rheb/mTOR pathway in ischemic stroke. We also investigated how these two elements interact with each other and contribute to injuries resulting from ischemic stroke. MATERIAL AND METHODS We used both a middle cerebral artery occlusion rat model in vivo and an oxygen-glucose deprivation cell model in vitro to simulate the onset of ischemic stroke. miR-155 mimics, miR-155 inhibitors, and Rheb siRNA were transfected to alter the expression of miR-155 and Rheb. Infarct sizes were measured using magnetic resonance imaging (MRI) and triphenyltetrazolium chloride (TTC) staining; cell apoptosis rates were calculated using Annexin V-FITC/PI staining and flow cytometry. Levels of miR-155, Rheb, mTOR, and S6K were examined by RT-PCR, immunofluorescence, and western blot. We performed a luciferase activity assay so that the association between miR-155 and Rheb could be fully assessed. RESULTS We demonstrated that miR-155 bound the 3'-UTR of Rheb and suppressed Rheb expression. As suggested by animal models, significant cerebral infarct volumes and cell apoptosis were induced by increased expression of miR-155 and decreased expression of Rheb, mTOR, and p-S6K (P<0.05). miR-155 inhibitors exhibited protective effects on ischemic stroke, including down-regulation of infarction size in cerebral tissues in vivo and reduced apoptosis of BV2 cells in vitro with increased expression of Rheb, mTOR and p-S6K (P<0.05). These protective effects could be substantially antagonized by the transfection of Rheb siRNA (P<0.05). CONCLUSIONS Inhibition of miR-155 may play protective roles in ischemic stroke by phosphorylating S6K through the Rheb/mTOR pathway.
Insights
Inhibition of microRNA-155 (miR-155) protects against ischemic stroke by regulating the Rheb/mTOR pathway. This finding offers a potential therapeutic target for stroke treatment.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Ischemic stroke remains a leading cause of disability and mortality worldwide.
- The molecular mechanisms underlying ischemic stroke injury are complex and not fully understood.
- MicroRNA-155 (miR-155) and the Rheb/mammalian target of rapamycin (mTOR) pathway have been implicated in cellular stress responses.
Purpose of the Study:
- To investigate the role of miR-155 and the Rheb/mTOR pathway in ischemic stroke.
- To elucidate the interaction between miR-155 and Rheb/mTOR in stroke-related injury.
- To evaluate the therapeutic potential of targeting miR-155 in ischemic stroke.
Main Methods:
- Utilized middle cerebral artery occlusion (MCAO) rat model and oxygen-glucose deprivation (OGD) cell model.
- Manipulated miR-155 and Rheb expression using mimics, inhibitors, and small interfering RNA (siRNA).
- Assessed infarct volume (MRI, TTC staining), cell apoptosis (Annexin V-FITC/PI, flow cytometry), and protein/gene expression (RT-PCR, immunofluorescence, Western blot, luciferase assay).
Main Results:
- miR-155 directly targets and suppresses Rheb expression.
- Elevated miR-155 and reduced Rheb, mTOR, and p-S6K expression exacerbated infarct size and cell apoptosis in vivo and in vitro.
- Inhibition of miR-155 demonstrated protective effects, reducing infarct volume and apoptosis, and increasing Rheb, mTOR, and p-S6K levels.
- The protective effects of miR-155 inhibition were diminished by Rheb siRNA, confirming the pathway's involvement.
Conclusions:
- miR-155 promotes ischemic stroke injury by suppressing Rheb and consequently inhibiting the Rheb/mTOR/S6K pathway.
- Inhibiting miR-155 offers a potential therapeutic strategy for ischemic stroke.
- Targeting miR-155 may protect brain tissue by restoring Rheb/mTOR pathway activity.
