lncRNA BDNF-AS靶向miR-375调控氧化型低密度脂蛋白诱导的动脉粥样硬化模型细胞损伤实验研究  

Experimental study on long non-coding RNA brain-derived neurotrophic factor antisense targeting miR-375 to regulate oxidized low density lipoprotein-induced cell damage in atherosclerosis model

作　　者：孙艳 田士宏 龚芳 Sun Yan;Tian Shihong;Gong Fang(Department of Neurology,Zaozhuang Mining Group Central Hospital,Zaozhuang 277000,China;Department of Cardiology,Affiliated Hospital of Hubei University of Arts and Sciences(Xiangyang Central Hospital),Xiangyang 441021,China)

机构地区：[1]山东国欣颐养集团枣庄中心医院神经内科,枣庄277000 [2]湖北文理学院附属医院(襄阳市中心医院)心内科,441021

出　　处：《心脑血管病防治》2023年第7期25-28,53,共5页CARDIO-CEREBROVASCULAR DISEASE PREVENTION AND TREATMENT

基　　金：湖北省自然科学基金(WJ2015Q037)。

摘　　要：目的探讨长链非编码RNA脑源性神经营养因子反义因子(lncRNA BDNF-AS)对氧化型低密度脂蛋白(ox-LDL)诱导的动脉粥样硬化模型细胞损伤的影响及其对miR-375的靶向调控作用。方法体外培养人脐静脉内皮细胞(HUVECs),分别将lncRNA BDNF-AS小分子干扰RNA与miR-375寡核苷酸模拟物及其阴性对照、lncRNA BDNF-AS小分子干扰RNA si-lncRNA BDNF-AS与miR-375特异性寡核苷酸抑制剂阴性对照、si-lncRNA BDNF-AS与miR-375特异性寡核苷酸抑制剂转染至HUVECs,并用ox-LDL处理24 h;仅使用ox-LDL处理的细胞为ox-LDL组,正常细胞为Con组。采用qRT-PCR法检测lncRNA BDNF-AS、miR-375的表达量;利用试剂盒检测丙二醛(MDA)的含量与超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性;流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测lncRNA BDNF-AS、miR-375的靶向关系。结果与Con组比较,ox-LDL组lncRNA BDNF-AS的表达水平升高(t=26.180,P<0.01),miR-375的表达水平降低(t=32.413,P<0.01);ox-LDL诱导的HUVECs中MDA含量、凋亡率升高(t=27.588、26.806,P<0.01),SOD、CAT的活性降低(t=18.925、37.887,P<0.01)。转染lncRNA BDNF-AS小分子干扰RNA后,lncRNA BDNF-AS的表达水平、MDA含量、凋亡率降低(t=17.220、15.527、18.930,P<0.01),SOD、CAT的活性升高(t=12.570、17.202,P<0.01)。双荧光素酶报告实验证实lncRNA BDNF-AS可靶向结合miR-375;转染miR-375寡核苷酸模拟物可明显降低MDA的含量及凋亡率(t=11.945、20.881,P<0.01),提高SOD、CAT的活性(t=15.859、13.102,P<0.05)。共转染si-lncRNA BDNF-AS和anti-miR-375后,MDA含量、凋亡率升高(t=10.712、15.218,P<0.01),SOD、CAT的活性降低(t=7.961、10.773,P<0.01)。结论抑制lncRNA BDNF-AS表达可上调miR-375的表达而抑制ox-LDL诱导HUVECs氧化应激及细胞凋亡,从而减轻动脉粥样硬化模型细胞损伤。Objective To investigate the effect of long non-coding RNA brain-derived neurotrophic factor antisense(lncRNA BDNF-AS)on oxidized low density lipoprotein(ox-LDL)-induced cell damage in an atherosclerosis model and its targeted regulation of miR-375.Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro.lncRNA BDNF-AS small interfering RNA(siRNA),miR-375 oligonucleotide mimics and their negative controls,lncRNA BDNF-AS siRNA and the negative control of miR-375 specific oligonucleotide inhibitor,lncRNA BDNF-AS siRNA and miR-375 specific oligonucleotide inhibitor were transfected into HUVECs,respectively.The cells were then treated with ox-LDL for 24 hours.Cells treated only with ox-LDL were the ox-LDL group,while normal cells were the Con group.qRT-PCR was used to detect the expression levels of lncRNA BDNF-AS and miR-375.The content of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)and catalase(CAT)were measured using assay kits.The apoptosis rate of the cells was measured by flow cytometry.The dual-luciferase reporter experiment was used to detect the targeting relationship between lncRNA BDNF-AS and miR-375.Results Compared with the Con group,the expression level of lncRNA BDNF-AS increased(t=26.180,P<0.01)and the expression level of miR-375 decreased(t=32.413,P<0.01)in the ox-LDL group.The content of MDA and the apoptosis rate in ox-LDL-induced HUVECs increased(t=27.588,26.806;P<0.01),and the activity of SOD and CAT decreased(t=18.925,37.887;P<0.01).After transfection with lncRNA BDNF-AS siRNA,the expression level of lncRNA BDNF-AS,MDA content,and apoptosis rate decreased(t=17.220,15.527,18.930;P<0.01),and the activity of SOD and CAT increased(t=12.570,17.202;P<0.01).The dual-luciferase reporter experiment confirmed that lncRNA BDNF-AS can targeted bind to miR-375.Transfection with miR-375 oligonucleotide mimics significantly decreased the content of MDA and the apoptosis rate(t=11.945,20.881;P<0.01),and increased the activity of SOD and CAT(t=15.859,13.102;P<0.05).After

关 键 词：长链非编码RNA脑源性神经营养因子反义因子 miR-375 氧化型低密度脂蛋白 动脉粥样硬化 氧化应激 凋亡 

分 类 号：R543.5[医药卫生—心血管疾病]