miR-188-5p在N2a细胞氧糖剥夺-复糖复氧损伤中的作用:与SENP3的关系

Role of miR-188-5p in oxygen-glucose deprivation and restoration injury to mouse N2a cells:relationship with SENP3

目的评价miR-188-5p在N2a细胞氧糖剥夺-复糖复氧(OGD/R)损伤中的作用及其与SUMO特异性蛋白酶3(SENP3)的关系。方法小鼠N2a细胞采用随机数字表法分为5组(n=23):对照组(C组)、OGD/R组、NC组、转染miR-188-5p激动剂组(M组)和转染miR-188-5p抑制剂组(I组)。C组细胞常规培养,NC组、M组和I组分别转染试剂miR-188-5p阴性对照miRNA、激动剂及抑制剂后。氧糖剥夺3 h后复糖复氧制备N2a细胞氧糖剥夺-复糖复氧损伤模型,于复糖复氧后24 h时采用CCK-8法检测细胞活力,检测LDH漏出量,采用RT-qPCR法检测miR-188-5p和SENP3 mRNA表达,采用Western blot法检测SENP3表达。采用双荧光素酶实验检测miR-188-5p与SENP3 mRNA的靶向关系。结果与C组比较,其余4组细胞活力降低,LDH漏出量增加,SENP3及其mRNA表达上调,OGD/R组和I组miR-188-5p表达下调,M组miR-188-5p表达上调(P<0.05或0.01);与OGD/R组比较,I组细胞活力降低,LDH漏出量增加,SENP3及其mRNA表达上调,miR-188-5p表达下调,M组细胞活力增加,LDH漏出量下降,SENP3及其mRNA表达下调,miR-188-5p表达上调(P<0.05或0.01)。双荧光素酶实验报告表明:miR-188-5p直接作用于SENP3。结论miR-188-5p参与了N2a细胞氧糖剥夺-复糖复氧损伤的过程,与靶向下调SENP3表达有关。

Objective To evaluate the role of miR-188-5p in oxygen-glucose deprivation and restoration(OGD/R)injury to mouse neuroblastoma(N2a)cells and its relationship with small ubiquitin-like modifier-specific proteases 3(SENP3).Methods N2a cells were cultured and divided into 5 groups(n=23 each)using a random number table method:control group(group C),OGD/R group,group NC,transfection of mir-188-5p agonist group(group M)and transfection of mir-188-5p inhibitor group group(group I).Cells in group C were cultured routinely.Cells in group NC,group M and group I were transfected with mir-188-5p negative control miRNA,agonist and inhibitor,respectively.N2a cells were subjected to OGD for 3 h followed by restoration of oxygen-glucose supply to establish the model of OGD/R injury.At 24 h of oxygen-glucose restoration,the cell viability was recorded by the cell counting kit-8 assay,the amount of lactic dehydrogenase(LDH)released was detected,the expression of miR-188-5p and SENP3 mRNA was detected by quantitative real-time polymerase chain reaction,and SENP3 expression was determined by Western blot.The targeting relationship between miR-188-5p and SENP3 mRNA was detected using dual luciferase reporter assay.Results Compared with group C,the cell viability was significantly decreased,amount of LDH released was increased,and expression of SENP3 and its mRNA was up-regulated in the other 4 groups,miR-188-5p expression was down-regulated in OGD/R and I groups,and miR-188-5p expression was up-regulated in group M(P<0.05 or 0.01).Compared with group OGD/R,the cell viability was significantly decreased,amount of LDH released was increased,and expression of SENP3 and its mRNA was up-regulated,and miR-188-5p expression was down-regulated in group I,and the cell viability was increased,amount of LDH released was decreased,expression of SENP3 and its mRNA was down-regulated,and miR-188-5p expression was up-regulated in group M(P<0.05 or 0.01).The dual luciferase reporter assay showed that miR-188-5p could act directly on SENP3.Conclusion miR-188-5p is involved in OGD/R injury,which is associated with targeted down-regulation of SENP3 expression in N2a cells.