CircCDC14A沉默保护星形胶质细胞免受OGD/R诱导的损伤作用机制分析

Mechanism analysis of CircCDC14A silencing to protect astrocytes from OGD/R induced injury

目的探讨CirCDC14A沉默通过调节miR-153-3p/JAK1轴保护星形胶质细胞免受氧糖剥夺/复糖复氧(OGD/R)诱导的损伤机制。方法体外培养新生大鼠脑皮质星形胶质细胞,采用CCK-8法法检测各组细胞活力;采用Annexin V-FITC/PI染色检测细胞凋亡情况;采用Western blot法检测各组p53、Bax、Bcl-2蛋白表达水平,并采用双荧光素酶报告基因实验分析CirCDC14A与miR-153-3p、miR-153-3p与JAK1的关系。结果随着时间的延长,细胞活性呈上升趋势;pcDNA3.0 CirCDC14A+si JAK1组48 h及72 h细胞活力低于si-NC组、OGD/R组、OGD/R+Si+Con mimcs组(P<0.05);与si-NC组相比,OGD/R组、OGD/R+Si+Con mimcs组、pcDNA3.0-CirCDC14A+si JAK1+miR-153-3p mimcs组细胞凋亡明显增加,且pcDNA3.0-CirCDC14A+si JAK1+miR-153-3p mimcs组细胞凋亡高于GD/R+Si+Con mimcs组(P<0.05);Western blot结果表明:pcDNA3.0-CirCDC14A+si JAK1+miR-153-3p mimcs组p53、Bax、Bcl-2蛋白表达水平低于OGD/R组、OGD/R+Si+Con mimcs组和si-NC组(P<0.05);miR-153-3p mimic能降低CirCDC14A WT、JAK1-WT的荧光素酶活性(P<0.05);pcDNA3.0-CirCDC14A+si JAK1+miR-153-3p mimcs组miR-153-3p水平低于于其余3组(P<0.05),JAK1蛋白水平高于其余3组(P<0.05)。结论CirCDC14A基因沉默可能通过调控miR-153-3p/JAK1轴抑制OGD/R诱导的大鼠星形胶质细胞凋亡,引起细胞活力及p53、Bax、Bcl-2蛋白表达水平降低。

Objective To investigate the mechanism of CirCDC14A silencing to protect astrocytes from oxygen-glucose deprivation/glyco-reoxygenation(OGD/R)-induced injury by regulating the miR-153-3p/JAK1 axis.Methods Cerebral cortical astrocytes of neonatal rats were cultured in vitro,and the cell viability was detected by CCK-8 method;annexin V-FITC/PI staining was used to detect cell apoptosis.Western blot method was used to detect the protein expression levels of p53,Bax and Bcl-2 in each group,and the relationship between CirCDC14A and miR-153-3p,miR-153-3p and JAK1 was analyzed by dual-luciferase reporter gene assay.Results The cell viability increased with time;the cell viability of pcDNA3.0 CirCDC14A+si JAK1 group at 48 h and 72 h was lower than that of si-NC group,OGD/R group and OGD/R+Si+Con mimcs group(P<0.05);compared with si-NC group,the apoptosis of OGD/R group,OGD/R+Si+Con mimcs group,pcDNA3.0-CirCDC14A+si JAK1+miR-153-3p mimcs group increased significantly,and pcDNA3.0-CirCDC14A+si JAK1+miR-153-3p mimcs group had higher apoptosis than OGD/R+Si+Con mimcs group(P<0.05);Western blot results showed that:pcDNA3.0-CirCDC14A+si JAK1+miR,the protein expression levels of p53,Bax and Bcl-2 in the miR-153-3p mimcs group were lower than those in the OGD/R group,OGD/R+Si+Con mimcs group and si-NC group(P<0.05).Reduced the luciferase activity of CirCDC14A WT and JAK1-WT(P<0.05);the level of miR-153-3p in the pcDNA3.0-CirCDC14A+si JAK1+miR-153-3p mimcs group was lower than that in the other three groups(P<0.05).The JAK1 protein level was higher than the other three groups(P<0.05).Conclusion CirCDC14A gene silencing may inhibit OGD/R-induced apoptosis of rat astrocytes by regulating the miR-153-3p/JAK1 axis,resulting in decreased cell viability and protein expression levels of p53,Bax and Bcl-2.