摘要
目的探讨环状RNA染色质重塑因子(Circular RNA bromodomain PHD finger transcription factor,circBPTF)对过氧化氢(H2O2)诱导的神经细胞氧化应激、凋亡的影响及其对微小RNA-107(microRNA-107,miR-107)的调控作用。方法体外培养人神经母细胞瘤细胞(Human neuroblastoma cells,SK-NSH),采用H2O2诱导SK-N-SH细胞建立细胞氧化损伤模型;siRNA阴性对照(siRNA negative control,si-NC),circBPTF小分子干扰RNA(circBPTF small interfering RNA,si-circBPTF)、阴性对照mimic NC序列(Negative control mimic NC sequence,miR-NC),miR-107寡核苷酸模拟物(miR-107 oligonucleotide mimic,miR-107 mimics),si-circBPTF与miRNA阴性对照(miRNA negative control,anti-miR-NC),si-circBPTF与miR-107特异性寡核苷酸抑制剂(miR-107 specific oligonucleotide inhibitor,anti-miR-107)转染至H2O2诱导的SK-N-SH细胞;采用实时定量PCR(Quantitative real-time reverse transcription PCR,qRT-PCR)法检测细胞中circBPTF,miR-107的相对表达水平;试剂盒检测乳酸脱氢酶(Lactate dehydrogenase,LDH)释放量与丙二醛(Malondialedhyde,MDA)、超氧化物歧化酶(Superoxide dismutase,SOD)的水平;采用流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测circBPTF与miR-107的靶向关系;Western blot法检测B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bcl-2-associated X protein,Bax)蛋白相对表达水平。结果H2O2诱导的SK-N-SH细胞中circBPTF的相对表达水平升高(P<0.05),miR-107的相对表达水平降低(P<0.05),LDH释放量与MDA的水平升高(P<0.05),凋亡率及Bax蛋白相对表达水平升高(P<0.05),Bcl-2蛋白相对表达水平降低(P<0.05);转染si-circBPTF或转染miR-107 mimics可明显降低H2O2诱导的SK-N-SH细胞中LDH释放量与MDA的水平及凋亡率(P<0.05),SOD的水平及Bcl-2蛋白相对表达水平升高(P<0.05),还可降低Bax蛋白相对表达水平(P<0.05);双荧光素酶报告实验证实circBPTF可靶向结合miR-107;共转染si-circBPTF与anti-miR-107可明显逆转转染si-cir
Objective To explore the effect of circBPTF onoxidative stress and apoptosis of neurons induced by hydrogen peroxide(H_(2)O_(2))and its regulatory effect on miR-107.Methods Human neuroblastoma cells SK-N-SH were cultured in vitro,and SK-N-SH cells were induced by H_(2)O_(2)to establish a celloxidativedamage model.si-NC,si-circBPTF,miR-NC,miR-107 mimics,si-circBPTF andanti-miR-NC,si-circBPTF and anti-miR-107 were transfected into SK-N-SH cells induced by H_(2)O_(2).The qRT-PCR method was used to detect the expression of circBPTF and miR-107in the cells.The kit was used to detect the release of LDH and the content of MDA and SOD.Flow cytometry was used to detect the apoptosis rate.The dual luciferase reporter experiment detects the targeting relationship between circBPTF and miR-107.Western blot was used to detect the expression of Bcl-2 and Bax protein.Results The protein expressionof circBPTF and Bax,the release of LDH,the content of MDA and apoptosis rate were significantly increased in SK-N-SH cells induced by H_(2)O_(2)(P<0.05).On the contrary,the expression of miR-107 and the protein level of Bcl-2 was significantly decreased(P<0.05).In addition,transfection ofsi-circBPTF or miR-107 mimics could significantly reduce LDH release,MDA level,Bax protein expression and apoptotic rate of SK-N-SH cells induced by H_(2)O_(2)(P<0.05).It also significantly increased SOD content and Bcl-2 protein level(P<0.05).The dual luciferase report experiment furtherly confirmed that circBPTF could target miR-107.Co-transfection of si-circBPTF and anti-miR-107 could significantly reverse the effect of transfection of si-circBPTF on apoptosis and oxidative stress of SK-N-SH cells induced by H_(2)O_(2).Conclusion Inhibiting the expression of circBPTF could up-regulate miR-107 and inhibit H_(2)O_(2)-induced oxidative stress and apoptosis of SK-N-SH cells,thereby reducing oxidative damage of neurons.
作者
黄本艳
刘群会
周振东
刘美霞
贾敏
李文静
Huang Benyan;Liu Qunhui;Zhou Zhendong(Department of Neurology,Enshi Central Hospital,Enshi Prefecture Hubei Province 445000)
出处
《卒中与神经疾病》
2021年第4期383-388,共6页
Stroke and Nervous Diseases
基金
湖北省自然科学基金计划项目(No.2014CFB424)。