LncRNA ANRIL对缺氧/复氧诱导的H9c2心肌细胞焦亡及NLRP3炎性体通路的影响  

The effects of LncRNA ANRIL on hypoxia/reoxygenation-induced H9c2 cardiomyocyte pyrolysis and NLRP3 inflammasome pathway

作　　者：谷庆波 张科成 于爱华 GU Qing-bo;ZHANG Ke-cheng;YU Ai-hua(Department of Cardiovascular Medicine,Yingkou Hos-pital of Traditional Chinese Medicine,Yingkou,Liaoning Province 115099,China)

机构地区：[1]营口市中医院心血管内科,辽宁营口115099

出　　处：《解剖学研究》2022年第1期12-17,共6页Anatomy Research

摘　　要：目的探讨长链非编码RNA(LncRNA)的INK4基因座中反义非编码RNA(ANRIL)对缺氧/复氧(A/R)诱导的H9c2心肌细胞焦亡及核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性体通路的影响。方法qRT-PCR检测LncRNA ANRIL在A/R诱导的H9c2细胞中的表达;通过慢病毒转染构建抑制LncRNA ANRIL的H9c2心肌细胞系,分组为:空白组、si-NC组、si-ANRIL组,转染24 h后,利用荧光显微镜观察荧光变化;qRT-PCR检测LncRNA ANRIL表达以验证转染结果;将慢病毒转染成功的H9c2心肌细胞进行缺氧24 h复氧6 h处理,并分组为,正常对照组(Ctrl组,细胞正常培养,未经任何处理)、A/R组、si-NC+A/R组、si-ANRIL+A/R组,免疫荧光法检测各组细胞中caspase-1蛋白表达;ELISA法检测各组细胞上清液中IL-1β和IL-18的含量;Western blot检测各组细胞中NLRP3、凋亡相关斑点样蛋白(ASC)的表达。结果 LncRNA ANRIL在A/R诱导的H9c2细胞中的表达水平显著高于正常细胞;荧光显微镜可观察到转染慢病毒的H9c2细胞呈现出绿色荧光,且si-ANRIL组H9c2细胞中ANRIL表达水平显著低于空白组和si-NC组(P<0.05),成功构建抑制ANRIL的H9c2心肌细胞系;与Ctrl组比较,A/R组H9c2细胞中IL-1β和IL-18含量、caspase-1、NLRP3、ASC蛋白表达水平显著升高(P<0.05);与A/R组和si-NC+A/R组比较,siANRIL+A/R组H9c2细胞中IL-1β和IL-18含量、caspase-1、NLRP3、ASC蛋白表达水平显著降低(P<0.05)。结论沉默LncRNA ANRIL可能通过抑制NLRP3炎性体通路来改善A/R诱导的H9C2心肌细胞焦亡。Objective To investigate the effects of antisense non-coding RNA in the INK4 locus(ANRIL)of long non-coding RNA(LncRNA)on hypoxia/reoxygenation(A/R)-induced H9 c2 cardiomyocyte pyrolysis and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome pathway. Methods QRT-PCR was used to detect the expression of LncRNA ANRIL in H9 c2 cells induced by A/R;the H9 c2 cardiomyocyte cell line inhibiting LncRNA ANRIL was constructed by lentiviral transfection and divided into:blank group,si-NC group and si-ANRIL group. After 24 hours of transfection,a fluorescence microscope was used to observe changes in fluorescence;qRT-PCR was used to detect the expression of LncRNA ANRIL to verify the transfection results;the H9 c2 cardiomyocytes successfully transfected with lentivirus were treated with hypoxia for 24 h and reoxygenation for 6 h,and grouped into normal control group(Ctrl group,cells were cultured normally,without any treatment),A/R group,si-NC+A/R group,si-ANRIL+A/R group,immunofluorescence method was used to detect the expression of caspase-1 protein in each group of cells;ELISA method was used to detect the contents of interleukin(IL)-1β and IL-18 in the cell supernatant of each group;Western blot was used to detect the expression of NLRP3 and apoptosis-related dot-like protein(ASC)in each group of cells. Results The expression level of LncRNA ANRIL in H9 c2 cells induced by A/R was significantly higher than that in normal cells;fluorescence microscopy showed that H9 c2 cells transfected with lentivirus showed green fluorescence,and the expression level of ANRIL in H9 c2 cells in the si-ANRIL group was significantly lower than that in the blank group and si-NC group(P<0.05),the H9 c2 cardiomyocyte cell line inhibiting ANRIL was successfully constructed;compared with the Ctrl group,the contents of IL-1βand IL-18,and the expression levels of caspase-1,NLRP3,and ASC proteins in H9 c2 cells in the A/R group were significantly increased(P<0.05);compared with the A/R group and the si-NC+A/R g

关 键 词：长链非编码RNA的INK4基因座中反义非编码RNA 缺氧/复氧 H9C2细胞 细胞焦亡 核苷酸结合寡聚化结构域样受体蛋白3炎性体通路 

分 类 号：R542.22[医药卫生—心血管疾病]