Wang He, Yu Qian, Zhang Zai-Li, Ma Hong, Li Xiao-Qian
Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang, 110001 Liaoning, China.
Department of Thoracic Surgery, Fourth Affiliated Hospital, China Medical University, Shenyang, 110032 Liaoning, China.
Oxid Med Cell Longev. 2020 Dec 10;2020:2616871. doi: 10.1155/2020/2616871. eCollection 2020.
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 and IR models.
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 Ca concentration and mean fluorescence intensity . 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.
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 Ca 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 . 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.
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.
缺血再灌注（IR）损伤后神经元存活是运动功能预后的主要决定因素。基于微小RNA（miR）的基因治疗近来受到关注。我们之前的工作探索了miR-137-3p在体内和体外IR模型中调节神经元凋亡的机制。
在IR后12小时内检测IR诱导的运动功能障碍以及脊髓钙蛋白酶（CAPN）亚型的表达和亚细胞定位。通过miR微阵列分析鉴定包括miR-137-3p在内的失调miR，并通过PCR进行确认。荧光素酶测定证实CAPN-2是miR-137-3p的相应靶标，并通过鞘内注射合成miR来评估它们对运动功能的调节。通过细胞内钙浓度和平均荧光强度测量CAPN-2活性。通过流式细胞术和TUNEL测定检测神经元凋亡。在用特异性抑制剂和miR转染后，通过ELISA和蛋白质印迹评估p35、p25、Cdk5和半胱天冬酶-8的活性。
IR诱导的运动功能障碍时间进程与CAPN-2蛋白表达上调密切相关，CAPN-2主要定位于神经元。荧光素酶测定证实了miR-137-3p/CAPN-2相互作用。即使与重组大鼠钙蛋白酶-2（rr-CALP2）注射联合使用，miR-137-3p模拟物也能显著改善IR诱导的运动功能障碍并降低CAPN-2表达，而miR-137-3p抑制剂则逆转了这些作用。当细胞暴露于氧-葡萄糖剥夺和再灌注（OGD/R）并转染合成miR时，观察到细胞内钙浓度、CAPN-2表达和CAPN-2活性有类似变化。此外，双荧光显示CAPN-2、p35、p25和半胱天冬酶-8在神经元中的定位相同。在用miR-137-3p模拟物、roscovitine（一种Cdk5抑制剂）或Z-IETD-FMK（一种半胱天冬酶-8抑制剂）转染的细胞中，CAPN-由于细胞内钙浓度、CAPN-2表达和CAPN-2活性有类似变化。此外，双荧光显示CAPN-2、p35、p25和半胱天冬酶-8在神经元中的定位相同。在用miR-137-3p模拟物、roscovitine（一种Cdk5抑制剂）或Z-IETD-FMK（一种半胱天冬酶-8抑制剂）转染的细胞中，CAPN-2表达和活性的降低伴随着p35活性和蛋白表达的相反变化。相应地，上述处理导致神经元存活率高于未处理的神经元，表现为凋亡细胞百分比以及p25、Cdk5、半胱天冬酶-8和半胱天冬酶-3蛋白表达降低。
miR-137-3p/CAPN-2相互作用在IR损伤期间调节神经元凋亡，可能是通过抑制CAPN-2，从而导致p35裂解并抑制随后的p25/Cdk5和半胱天冬酶-8过度激活。
