Neuroprotective Effects of lncRNA EPB41L4A-AS1 Against Ischemic Stroke Injury Through Regulating Microglial State Transition and Ferroptosis via miR-214-3p/GPX4 Axis.

EPB41L4A-AS1 was significantly downregulated in the serum of acute ischemic stroke (AIS) patients compared to controls.

• EPB41L4A-AS1 levels were measured by RT-qPCR in 120 AIS patients.
• Expression was also reduced in OGD/R (oxygen-glucose deprivation/reperfusion)-treated BV2 microglia, an in vitro model of ischemic injury.
• Lower EPB41L4A-AS1 levels predicted worse prognosis in AIS patients.

Elevated EPB41L4A-AS1 expression reduced pro-inflammatory markers and enhanced anti-inflammatory markers in OGD/R-injured BV2 microglial cells.

• Pro-inflammatory markers assessed included iNOS, IL-1β, and TNF-α, which were substantially reduced upon EPB41L4A-AS1 overexpression.
• Anti-inflammatory markers Arg1 and IL-10 were enhanced with elevated EPB41L4A-AS1 expression.
• These findings indicate that EPB41L4A-AS1 promotes a shift from a pro-inflammatory to an anti-inflammatory microglial state.
• ELISA and western blot were used to assess microglial state transition markers.

Elevated EPB41L4A-AS1 expression improved iron overload and lipid peroxidation in OGD/R-injured BV2 cells, suggesting inhibition of ferroptosis.

• Iron overload and lipid peroxidation are hallmarks of ferroptosis, a form of regulated cell death.
• Both parameters were improved in OGD/R-injured BV2 cells upon EPB41L4A-AS1 overexpression.
• ELISA and western blot were employed to assess ferroptosis-related changes.

RIP, RNA pull-down, and luciferase reporter assays confirmed that EPB41L4A-AS1 specifically binds to miR-214-3p, and miR-214-3p directly targets GPX4.

• A combination of RNA immunoprecipitation (RIP), RNA pull-down, and luciferase reporter assays were employed to validate these molecular interactions.
• EPB41L4A-AS1 functions as a competing endogenous RNA (ceRNA) by sponging miR-214-3p.
• GPX4 (glutathione peroxidase 4) was identified as a downstream target of miR-214-3p.
• This confirms the EPB41L4A-AS1/miR-214-3p/GPX4 regulatory axis.

miR-214-3p mimic amplified iron overload, oxidative stress, and pro-inflammatory state in EPB41L4A-AS1-overexpressing BV2 cells under OGD/R conditions, and these effects were abrogated by restoring GPX4 expression.

• Functional rescue assays were conducted by co-transfecting miR-214-3p with GPX4 to validate the regulatory relationship in vitro.
• Introduction of miR-214-3p mimic reversed the protective effects of EPB41L4A-AS1 overexpression, increasing iron overload and oxidative stress.
• miR-214-3p mimic also shifted BV2 cells back toward a pro-inflammatory state under OGD/R conditions.
• Restoring GPX4 expression abrogated all observed detrimental effects of miR-214-3p mimic, confirming GPX4 as the functional downstream effector.