CircHIPK3低表达调控miR-124/STAT3轴抑制Aβ诱导的海马神经元凋亡的机制研究

Low expression of circHIPK3 regulates the miR-124/STAT3 axis and inhibits Aβ-induced apoptosis of hippocampal neurons

目的探讨环状RNA同源域相互作用蛋白激酶3(circ HIPK3)低表达对β-淀粉样蛋白(Aβ)诱导的海马神经元凋亡的影响。方法采用生物信息学软件预测和双荧光素酶报告基因实验验证微小RNA-124(mi R-124)与HIPK3、信号转导和转录激活因子3(STAT3)的靶向关系;将体外培养的原代海马神经元分为对照组(正常培养)、Aβ组(给予Aβ刺激)、Aβ+si STAT3-NC组(转染si STAT3-NC后给予Aβ刺激)、Aβ+si HIPK3组(转染si HIPK3后给予Aβ刺激)、Aβ+si HIPK3+antimi R-NC组(共转染si HIPK3和inhibitor-NC后给予Aβ刺激)、Aβ+si HIPK3+antimi R-124组(共转染si HIPK3和mi R-124 inhibitor后给予Aβ刺激)、Aβ+si HIPK3+antimi R-124+si STAT3-NC组(共转染si HIPK3、mi R-124 inhibitor和si STAT3-NC后给予Aβ刺激)和Aβ+si HIPK3+antimi R-124+si STAT3组(共转染si HIPK3、mi R-124 inhibitor和si STAT3后给予Aβ刺激)。采用实时荧光定量PCR(q RT-PCR)检测HIPK3和mi R-124表达水平,免疫印迹法(Western blot)检测STAT3蛋白表达水平,噻唑蓝(MTT)法和流式细胞仪分别检测海马神经元存活率和凋亡率,含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)活性检测试剂盒检测Caspase-3活性。结果生物信息学软件预测、双荧光素酶报告基因实验证实HIPK3可与mi R-124靶向结合;生物信息学软件预测、双荧光素酶报告基因实验和Western blot实验证实,mi R-124可靶向调控STAT3蛋白表达。与对照组比较,Aβ组海马神经元中HIPK3表达水平、STAT3蛋白表达水平、凋亡率和Caspase-3活性明显升高,而海马神经元存活率和mi R-124表达水平明显降低(P<0.05);与Aβ+si HIPK3-NC组比较,Aβ+si HIPK3组中上述各指标明显逆转(P<0.05);与Aβ+si HIPK3+antimi R-NC组比较,Aβ+si HIPK3+antimi R-124组中各指标呈明显变化(P<0.05),且趋势与Aβ+si HIPK3组相反;与Aβ+si HIPK3+antimi R-124+si STAT3-NC组比较,Aβ+si HIPK3+antimi R-124+si STAT3组中各指标呈明显变化(P<0.05),且趋势与Aβ+si HIPK3+antimi R-124组相反;而Aβ组与Aβ+si HIPK3-NC组之间、Aβ+si HIPK3组与Aβ+si HIPK3+antimi R-NC组之间以及Aβ+si HIPK3+antimi R-124组与Aβ+si HIPK3+antimi R-124+si STAT3-NC组之间各指标差异无统计学意义(P>0.05)。结论Circ HIPK3低表达可抑制Aβ诱导的海马神经元凋亡,其作用机制与调控mi R-124/STAT3轴有关。

Objective To explore the effect of low expression of circular RNA homeodomaininteractingproteinkinases 3(circ HIPK3)on the apoptosis of hippocampal neurons induced by Amyloidβ-protein(Aβ).Methods Bioinformatics software prediction and double luciferase reporter gene experiments were used to investigate the targeting relationships between micro RNA-124(mi R-124)with HIPK3 and signal transduction and activator of transcription3(STAT3).Primary hippocampal neurons were cultured in vitro and divided into the control group(normal cultured),the amyloidβ-protein(Aβ)group(Aβstimulation),the Aβ+si STAT3-NC group(Aβstimulation after transfection of si STAT3-NC),the Aβ+si HIPK3 group(Aβstimulation after transfection of si HIPK3),the Aβ+si HIPK3+antimi R-NC group(Aβstimulation after cotransfection of si HIPK3 and inhibitor-NC),the Aβ+si HIPK3+antimi R-124 group(Aβstimulation after cotransfection of si HIPK3 and mi R-124 inhibitor),the Aβ+si HIPK3+antimi R-124+si STAT3-NC group(Aβstimulation after cotransfection of si HIPK3,mi R-124 inhibitor and si STAT3-NC)and the Aβ+si HIPK3+antimi R-124+si STAT3 group(Aβstimulation after cotransfection of si HIPK3,mi R-124 inhibitor and si STAT3).The expression levels of HIPK3 and mi R-124 were detected by real-time fluorescence quantitative PCR(q RT-PCR),and expression level of STAT3 protein was detected by Western blot.Survival and apoptosis rates of hippocampal neurons were measured by the thiazole blue(MTT)method and flow cytometry,respectively.Caspase-3 activity was detected by Cysteine-containing aspartate proteolytic enzyme 3(Caspase-3)activity detection kit.Results Bioinformatics and double luciferase reporter gene experiments showed that HIPK3 targeted mi R-124.Bioinformatics,double luciferase reporter gene experiments and Western blot demonstrated that mi R-124 targeted STAT3 protein expression.Compared with the control group,expression levels of HIPK3 and STAT3 protein,and the apoptosis rate and caspase-3 activity in hippocampal neurons were significantly higher in the Aβgroup,whereas the survival rate of neurons and expression level of mi R-124 were significantly lower(P<0.05).Compared with those in the Aβ+si STAT3-NC group,the above indexes were significantly reversed in the Aβ+si HIPK3 group(P<0.05).Compared with those in Aβ+si HIPK3+antimi R-NC group,the above indexes showed significant changes in the Aβ+si HIPK3+antimi R-124 group(P<0.05),and the trend was opposite to those in the Aβ+si HIPK3 group.Compared with the Aβ+si HIPK3+antimi R-124+si STAT3-NC group,the indexes of the Aβ+si HIPK3+antimi R-124+si STAT3 group were markedly changed(P<0.05),and the trend was opposite to those of the Aβ+si HIPK3+antimi R-124 group.There were no significant differences between the Aβand Aβ+si HIPK3-NC groups,the Aβ+si HIPK3 and Aβ+si HIPK3+antimi R-NC groups,or the Aβ+si HIPK3+antimi R-124+si STAT3-NC and Aβ+si HIPK3+antimi R-124+si STAT3 groups.Conclusions The low expression of circ HIPK3 can inhibit apoptosis of hippocampal neurons induced by Aβ,and its mechanism is related to regulation of the mi R-124/STAT3 axis.