摘要
目的:研究lncRNA GABPB1-AS1调控氧化应激适应性线粒体生成的关键调控因子PGC-1α和GABPB1的活化的作用机理。方法:人脐静脉内皮细胞(HUVEC)以200 μM的H2O2刺激3h构建氧化应激细胞模型,去除H2O2后继续培养,检测GABPB1-AS1在细胞应激后阶段的表达行为及其对PGC-1α和GABPB1的调控作用。1)qRT-PCR检测GABPB1-AS1和PGC-1α在去除H2O2后2h、4h、8h和16h表达情况,western bloting(WB)检测对应时段PGC-1α蛋白水平。转染GABPB1-AS1的siRNA和表达质粒,WB检测其对PGC-1α蛋白水平的影响。Tagertscan数据库预测miR-101与GABPB1-AS1和PGC-1α的结合关系,RNA pull down检测GABPB1-AS1对miR-101的亲和吸附作用,转染miR-101的mimic和inhibitor,WB检测其对PGC-1α蛋白水平的影响;2)qRT-PCR检测GABPB1-AS1和GABPB1在去除H2O2后2h、4h、8h、16和24h表达情况,WB检测对应时段GABPB1蛋白水平及过表达GABPB1-AS1后GABPB1蛋白水平。NCBI数据库预测并用RNA pull down和RNAprotection assay(RPA)验证GABPB1-AS1与GABPB1序列重叠位点、亲和吸附作用及其生理意义。结果:1)氧化应激诱导高表达的GABPB1-AS1在去除H2O2后逐渐降低至基线水平,对应时段PGC-1α蛋白水平显著增高;敲低和过表达GABPB1-AS1可以分别抑制和上调PGC-1α蛋白水平;miR-101在GABPB1-AS1和PGC-1α的3’UTR上均有结合位点,RNA pull down证明GABPB1-AS1亲和结合miR-101,过表达和敲除miR-101分别抑制和上调PGC-1α蛋白水平;2)氧化应激作用诱导GABPB1-AS1与GABPB1协同表达,而GABPB1蛋白变化则呈“U”曲线,过表达GABPB1-AS1下调GABPB1 mRNA及蛋白水平,GABPB1-AS1与GABPB1 mRNA的前端1~449 nt序列互补,RNA pull down和RPA实验证实二者在1~447 nt互补结合形成重叠二聚体结构,并保持GABPB1 mRNA稳定。结论:氧化应激诱导高表达的LncRNA GABPB1-AS1在应激适应阶段通过GABPB1-AS1/miR-101/PGC-1α轴,上调PGC-1α蛋白水平,促进应激适应性线粒体生成,GABPB1-AS1作为核质互作信号分子参与氧化�
Objective: To investigate the effect and mechanism of long non-coding RNA GABPB1-AS1 in regulating oxidative stress adaptive mitochondrial biogenesis by activating PGC-1α and GABPB1.Methods: The oxidative stress cell model was established by using human umbilical vein endothelial cells (HUVEC) which were stimulated with 200 μM H2O2 for 3 h,after removing H2O2,the expression of GABPB1-AS1 and its regulation of PGC-1α and GABPB1 during the phase of post-oxidative stress were detected.1) qRT- PCR was performed to detect the gene expression of GABPB1-AS1 and PGC-1α at 2 h,4 h,8 h and 16 h after the removal of H2O2,and western blotting (WB) was used to detect the protein level of PGC-1α at the corresponding time.Transfecting with siRNA and expression plasmid to knockdown and overexpress of GABPB1-AS1,then WB was used to detect its effect on the protein level of PGC-1α.To profile and analyze the miR-101’s binding to GABPB1-AS1 and PGC-1α's 3'UTR and its effects by utilizing the Tagertscan and TCGA databases,the RNA pull down trial was used to detect the affinity adsorption of GABPB1-AS1 on miR-101,and WB was used to detect the PGC-1α protein level after transfection with miR-101 mimic and inhibitor.2) qRT-PCR was performed to detect the expression of GABPB1-AS1 and GABPB1 at 2 h,4 h,8 h,16 h and 24 h after the removal of H2O2,and WB was used to detect the protein level of GABPB1 at the corresponding time.Overexpressing GABPB1-AS1,and then WB was performed to detect its effect on the protein level of GABPB1.To profile and analyze the overlapping sites,physically binding between GABPB1-AS1 and its sense sequence,the GeneCard and NCBI database were utilized as well as RNA pull down trial and PRA.Results: 1) The high expression of GABPB1-AS1 induced by oxidative stress gradually decreased to the baseline level after the removal of H2O2,the protein level but not mRNA level of PGC-1α was significantly increased.Knockdown of GABPB1-AS1 inhibited PGC-1α protein level,while GABPB1-AS1 overexpression reveled opposite
作者
徐思
李野
XU Si;LI Ye(School of Physical Education,Sichuan Normal University,Chengdu 610100,China)
出处
《体育科学》
CSSCI
北大核心
2019年第7期45-53,共9页
China Sport Science
