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Involvement of the miR-137-3p/CAPN-2 Interaction in Ischemia-Reperfusion-Induced Neuronal Apoptosis through
He Wang1, Qian Yu2, Zai-Li Zhang1
1Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang, 110001 Liaoning, China.
Background:
Neuron survival after ischemia-reperfusion (IR) injury is the primary determinant of motor function prognosis. MicroRNA- (miR-) based gene therapy has gained attention recently. Our previous work explored the mechanisms by which miR-137-3p modulates neuronal apoptosis in both in vivo and in vitro IR models.
Methods:
IR-induced motor dysfunction and spinal calpain (CAPN) subtype expression and subcellular localization were detected within 12 h post IR. Dysregulated miRs, including miR-137-3p, were identified by miR microarray analysis and confirmed by PCR. A luciferase assay confirmed CAPN-2 as a corresponding target of miR-137-3p, and their modulation of motor function was evaluated by intrathecal injection with synthetic miRs. CAPN-2 activity was measured by the intracellular Ca2+ concentration and mean fluorescence intensity in vitro. Neuronal apoptosis was detected by flow cytometry and TUNEL assay. The activities of p35, p25, Cdk5, and caspase-8 were evaluated by ELISA and Western blot after transfection with specific inhibitors and miRs.
Results:
The IR-induced motor dysfunction time course was closely associated with upregulated expression of the CAPN-2 protein, which was mainly localized in neurons. The miR-137-3p/CAPN-2 interaction was confirmed by luciferase assay. The miR-137-3p mimic significantly improved IR-induced motor dysfunction and decreased CAPN-2 expression, even in combination with recombinant rat calpain-2 (rr-CALP2) injection, whereas the miR-137-3p inhibitor reversed these effects. Similar changes in the intracellular Ca2+ concentration, CAPN-2 expression, and CAPN-2 activity were observed when cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R) and transfected with synthetic miRs in vitro. Moreover, double fluorescence revealed identical neuronal localization of CAPN-2, p35, p25, and caspase-8. The decrease in CAPN-2 expression and activity was accompanied by the opposite changes in p35 activity and protein expression in cells transfected with the miR-137-3p mimic, roscovitine (a Cdk5 inhibitor), or Z-IETD-FMK (a caspase-8 inhibitor). Correspondingly, the abovementioned treatments resulted in a higher neuron survival rate than that of untreated neurons, as indicated by decreases in the apoptotic cell percentage and p25, Cdk5, caspase-8, and caspase-3 protein expression.
Conclusions:
The miR-137-3p/CAPN-2 interaction modulates neuronal apoptosis during IR injury, possibly by inhibiting CAPN-2, which leads to p35 cleavage and inhibition of subsequent p25/Cdk5 and caspase-8 overactivation.
Insights
MicroRNA-137-3p protects neurons from ischemia-reperfusion injury by targeting calpain-2, improving motor function and reducing apoptosis. This microRNA therapy offers a promising avenue for treating neurological damage.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Neuron survival post-ischemia-reperfusion (IR) injury is critical for motor function recovery.
- MicroRNA (miR)-based gene therapy is a developing strategy for neurological conditions.
- Previous research identified miR-137-3p's role in modulating neuronal apoptosis in IR models.
Purpose of the Study:
- To investigate the therapeutic potential of miR-137-3p in mitigating IR-induced neuronal damage.
- To elucidate the molecular mechanisms underlying miR-137-3p's neuroprotective effects.
- To confirm the interaction between miR-137-3p and calpain-2 (CAPN-2) in the context of IR injury.
Main Methods:
- Assessed IR-induced motor dysfunction and spinal CAPN subtype expression.
- Utilized miR microarray and PCR to identify dysregulated miRs, focusing on miR-137-3p.
- Employed luciferase assays, intrathecal injections of synthetic miRs, and in vitro cell models (OGD/R) to confirm miR-137-3p/CAPN-2 interaction and functional effects.
- Analyzed neuronal apoptosis using flow cytometry and TUNEL assays.
- Quantified protein expression and activity of key molecules (p35, p25, Cdk5, caspase-8, caspase-3) via ELISA and Western blot.
Main Results:
- IR injury correlated with increased neuronal CAPN-2 expression and motor dysfunction.
- miR-137-3p mimic administration significantly improved motor function and reduced CAPN-2 levels, while the inhibitor reversed these effects.
- In vitro studies confirmed miR-137-3p's ability to decrease intracellular Ca2+ concentration and CAPN-2 activity.
- miR-137-3p treatment inhibited p35 cleavage, p25/Cdk5 activation, and caspase-8/caspase-3 mediated apoptosis, enhancing neuron survival.
Conclusions:
- The miR-137-3p/CAPN-2 axis is a key regulator of neuronal apoptosis in IR injury.
- Targeting CAPN-2 with miR-137-3p offers a potential therapeutic strategy to prevent neuronal death and improve outcomes after IR.
- This interaction influences downstream pathways involving p35 cleavage and caspase activation, highlighting a novel mechanism for neuroprotection.
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