circ_EFCAB2在癫痫细胞模型中的表达及其作用机制

Expression of circ_EFCAB2 in epileptic cell model and its mechanism

目的:探讨circ_EFCAB2在体外癫痫细胞模型中的表达,阐明其可能的作用机制。方法:人神经母细胞瘤LA-N-5细胞中构建无镁癫痫细胞模型(模型组),未经无镁细胞外液处理的细胞作为对照组。实时荧光定量PCR (RT-qPCR)法检测2组细胞中circ_EFCAB2 mRNA表达水平。将细胞分为核糖核酸酶(RNase) R消化组和RNase R未消化组,RNA酶消化实验和RT-qPCR法检测2组细胞中circ_EFCAB2和线性EFCAB2 mRNA表达水平。核质分离实验检测癫痫细胞中circ_EFCAB2 mRNA表达水平,CCK-8法检测2组细胞增殖能力,流式细胞术检测2组细胞凋亡率和不同细胞周期细胞百分率。结果:模型组细胞有自发高频率的痫样放电,提示癫痫细胞模型建立成功。RT-qPCR法检测,与对照组比较,模型组细胞中circ_EFCAB2 mRNA表达水平升高(P<0.05)。RNA酶消化实验和RT-qPCR法检测,与RNase R未消化组比较,RNase R消化组细胞中线性EFCAB2 mRNA表达水平明显降低(P<0.01)。核质分离实验检测,癫痫细胞的细胞质和细胞核中circ_EFCAB2 mRNA表达水平比较差异无统计学意义(P>0.05)。CCK-8法检测,转染72 h时,与对照组比较,模型组细胞增殖能力明显降低(P<0.01)。流式细胞术检测,与对照组比较,模型组细胞凋亡率明显升高(P<0.01);与对照组比较,模型组S期细胞百分率明显降低(P<0.01),G_(1)+G_(2)期细胞百分率明显升高(P<0.01)。结论:上调的circ_EFCAB2可能通过抑制细胞增殖、促进细胞凋亡和阻滞细胞周期等途径在癫痫的发病过程中发挥作用。

Objective:To discuss the expression of circs_EFCAB2 in epileptic cell model in vitro,and to clarify its possible mechanism.Methods:The magnesium-free epilepsy cell model(model group)was constructed in the human neuroblastoma LA-N-5 cells,and the cells untreated with magnesium-free extracellular fluid were regarded as control group.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression level of circ_EFCAB2 mRNA in the cells in two groups.The cells were didived into ribonuclease(RNase)R digestion group and RNase R undigestion group.RNA enzyme digestion experiment and RT-qPCR method were used to detect the expression levels of circ_EFCAB2 and linear EFCAB2 mRNA in the cells in two groups;nuclear cytoplasmic separation experiment was used to detect the expression levels of circle_EFCAB2 mRNA in the epileptic cells;CCK-8 method was used to detect the proliferation activities of the cells in two groups;flow cytometry was used to detect the apoptotic rate and percentages of the cells at different cell cycles in two groups.Results:The cells in model group showed spontaneous high-frequency epileptiform discharges,indicating the epilepsy cell model was successfully established.The RT-qPCR results showed that compared with control group,the expression level of circ_EFCAB2 mRNA in the cells in model group was increased(P<0.05).The RNA enzyme digestion experiment and RT-qPCR results showed that compared with RNase R undigestion group,the expression level of linear EFCAB2 mRNA in the cells in RNase R digestion group was decreased(P<0.01).The nuclear cytoplasmic separation experiment results showed that there was no statistically significant difference in the expression level of circ_EFCAB2 mRNA in the epileptic cells between the cytoplasm and nucleus of the epileptic cells(P>0.05).The CCK-8 results showed that at 72 h after transfection,compared with control group,the proliferation activity of the cells in model group was significantly decreased(P<0.01).The flow cytometry analysis results showed that compared with control group,the apoptotic rate of the cells in model group was significantly increased(P<0.01);compared with control group,the percentage of the cells at S phase in model group was significantly decreased(P<0.01),and the percentage of the cells at G_(1)+G_(2) phase was significantly increased(P<0.01).Conclusion:The upregulated circ_EFCAB2 may play a role in the pathogenesis of epilepsy by inhibiting the cell proliferation,promoting the apoptosis,and blocking the cell cycles.