Roles of the miR-137-3p/CAPN-2 gene pair in ischemia-reperfusion-induced neuronal apoptosis through modulation of p35 cleavage and subsequent caspase-8 overactivation

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. 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 distribution were detected within 12 h post IR. Dysregulated miRs, including miR-137-3p, were identified by miR microarray analysis and confirmed by PCR. Luciferase assay confirmed that CAPN-2 is a corresponding target of miR-137-3p, and their modulation of motor function was evaluated by intrathecal infection 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 lactate dehydrogenase (LDH) release. The activities of p35, p25, Cdk5 and caspase-8 were evaluated by ELISA and Western blotting after transfection with specific inhibitors and miRs.Results: The IR-induced motor dysfunction time course was closely associated with CAPN-2 protein upregulation, which was mainly distributed in neurons. The miR-137-3p/CAPN-2 gene pair was confirmed by luciferase assay. 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 miR-137-3p inhibitor reversed these effects. Similar changes were observed in the intracellular Ca2+ concentration and CAPN-2 expression and activity when cells were exposed to OGD/R and transfected with synthetic miRs in vitro. Moreover, double fluorescence revealed that CAPN-2, p35, p25 and caspase-8 were all identically distributed in neurons. The decrease in CAPN-2 expression and activity was accompanied by the opposite changes in p35 activity and protein expression in cells transfected with miR-137-3p mimic, roscovitine (a Cdk5 inhibitor) or Z-IETD-FMK (a caspase-8 inhibitor). Correspondingly, more surviving neurons were observed with the abovementioned treatments, indicated by a decrease in apoptotic cell percentage, LDH release and p25, Cdk5, caspase-8 and caspase-3 protein expression.Conclusions: The miR-137-3p/CAPN-2 gene pair functions to modulate neuronal apoptosis during IR injury, possibly through CAPN-2 inhibition leading to p35 cleavage and inhibition of subsequent p25/Cdk5 and caspase-8 overactivation.