Involvement of the miR-137-3p/CAPN-2 Interaction in Ischemia-Reperfusion-Induced Neuronal Apoptosis through Modulation of p35 Cleavage and Subsequent Caspase-8 Overactivation.
Oxid Med Cell Longev
; 2020: 2616871, 2020.
Article
em En
| MEDLINE
| ID: mdl-33456665
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Contexto em Saúde:
6_ODS3_enfermedades_notrasmisibles
Base de dados:
MEDLINE
Assunto principal:
Calpaína
/
Traumatismo por Reperfusão
/
Apoptose
/
MicroRNAs
/
Quinase 5 Dependente de Ciclina
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Caspase 8
/
Neurônios
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Oxid Med Cell Longev
Ano de publicação:
2020
Tipo de documento:
Article