摘要
目的:探讨环状 RNA OGDH(CircOGDH)通过调控 miR-195-5p/磷酸甘油酸变位酶 5(PGAM5)轴对氧糖剥夺/复糖复氧(OGD/R)诱导的小胶质(MG)细胞损伤的影响.方法:将体外培养的HMC3 细胞分为:NC组(正常培养)、OGD/R、si-NC 组、si-CircOGDH 组、mimic NC 组、miR-195-5p mimic组、si-CircOGDH+inhibitor NC组、si-CircOGDH+miR-195-5p inhibitor组.除NC 组外,剩余组在转染对应物质前构建 OGD/R 模型.qRT-PCR 法测定 CircOGDH、miR-195-5p、PGAM5 mRNA 表达水平;测定各组细胞增殖、凋亡、活性氧(ROS)及炎性因子水平;Western blot 检测 Cleaved caspase-3、Bax表达;双荧光素酶基因实验检测 CircOGDH 与 miR-195-5p、PGAM5 与 miR-195-5p 之间的靶向关系.结果:与NC组对比,OGD/R组miR-195-5p表达、OD450 值均降低,CircOGDH、PGAM5 mRNA表达、凋亡率、ROS及炎性因子水平、Cleaved caspase-3、Bax 蛋白表达均升高(P<0.05);对比 OGD/R 组和 si-NC组,si-CircOGDH 组 miR-195-5p 表达、OD450 值均升高,CircOGDH、PGAM5 mRNA 表达、凋亡率、ROS及炎性因子水平、Cleaved caspase-3、Bax蛋白表达均降低(P<0.05);miR-195-5p mimic 组分别与OGD/R组、mimic NC 组对比,CircOGDH 无显著差异(P>0.05),miR-195-5p 表达、OD450 值均升高,PGAM5 mRNA表达、凋亡率、ROS及炎性因子水平、Cleaved caspase-3、Bax 蛋白表达均降低(P<0.05);与si-CircOGDH+inhibitor NC组对比,si-CircOGDH+miR-195-5p inhibitor组CircOGDH差异不显著(P>0.05),miR-195-5p表达、OD450 值均降低,PGAM5 mRNA表达、凋亡率、ROS及炎性因子水平、Cleaved caspase-3、Bax 蛋白表达均升高(P<0.05).双荧光素酶基因实验显示 miR-195-5p 和 CircOGDH、PGAM5 均存在靶向关系.结论:沉默CircOGDH可促进OGD/R诱导的HMC3 细胞增殖,抑制其炎症反应和凋亡,可能与调控miR-195-5p/PGAM5 轴有关.
Abstract
Objective:To investigate the effect of circular RNA OGDH(CircOGDH)on microglia(MG)cell damage induced by oxygen-glucose deprivation/reoxygenation(OGD/R)by modulating the miR-195-5p/phosphoglycerate mutase 5(PGAM5)axis.Methods:HMC3 cells cultured in vitro were separated into:NC group(normal culture),OGD/R,si-NC group,si-CircOGDH group,mimic NC group,miR-195-5p mimic group,si-CircOGDH+inhibitor NC group,and si-CircOGDH+miR-195-5p inhibitor group.Except for the NC group,the remaining groups constructed OGD/R models before transfecting the corresponding sub-stances.QRT-PCR method was applied to determine the expression levels of CircOGDH,miR-195-5p,and PGAM5 mRNA.The levels of cell proliferation,apoptosis,reactive oxygen species(ROS),and inflammatory factors were measured in each group.Western blot was applied to detect the expression of Cleaved caspase-3 and Bax.Dual luciferase gene experiment was used to detect the targeting relationship between CircOGDH and miR-195-5p,and between PGAM5 and miR-195-5p.Results:Compared with the NC group,the expres-sion of miR-195-5p and OD450 value were reduced in the OGD/R group,the mRNA expression of CircOG-DH and PGAM5,apoptosis rate,ROS,and inflammatory cytokine levels,and the expression of Cleaved caspase-3 and Bax proteins were all elevated(P<0.05).Compared with the OGD/R group and the si-NC group,the expression of miR-195-5p and OD450 value were elevated in the si-CircOGDH group,the mRNA expression of CircOGDH and PGAM5,apoptosis rate,ROS and inflammatory cytokine levels,and the expres-sion of Cleaved caspase-3 and Bax proteins were all reduced(P<0.05).Compared with the OGD/R group and the mimic NC group,the miR-195-5p mimic group showed no significant difference in CircOGDH(P>0.05),while the expression of miR-195-5p and OD450 values were increased,the expression of PGAM5 mRNA,apoptosis rate,ROS and inflammatory cytokine levels,and the expression of Cleaved caspase-3 and Bax protein were all reduced(P<0.05).Compared with the si-CircOGDH+inhibitor NC group,the CircOG-DH of the si-CircOGDH+miR-195-5p inhibitor group was not significantly different(P>0.05),the expres-sion of miR-195-5p and OD450 values were decreased,the expression of PGAM5 mRNA,apoptosis rate,ROS and inflammatory cytokine levels,and the expression of Cleaved caspase-3 and Bax protein were all ele-vated(P<0.05).A dual luciferase gene experiment showed that miR-195-5p had a targeting relationship with CircOGDH and PGAM5.Conclusion:Silencing CircOGDH can promote the proliferation of HMC3 cells induced by OGD/R,inhibit their inflammatory response and apoptosis,and may be related to the regulation of miR-195-5p/PGAM5 axis.
基金项目
2024年度河北医学科学研究计划(20240045)
国家科技期刊平台
NSTL
万方数据