He Wang1, Qian Yu2, Zai-Li Zhang1, Hong Ma1, Xiao-Qian Li1. 1. Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang, 110001 Liaoning, China. 2. Department of Thoracic Surgery, Fourth Affiliated Hospital, China Medical University, Shenyang, 110032 Liaoning, China.
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.
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.
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