5′tRF-LysCTT对小鼠心肌细胞铁死亡的调控作用及其机制  

Regulatory effect of 5′tRF-LysCTT on ferroptosis in mouse cardiomyocytes and its mechanism

作　　者：赵炎 王凯[1] 程雪丽 王昆[1] ZHAO Yan;WANG Kai;CHENG Xueli;WANG Kun(Institute of Translational Medicine,Qingdao University,Qingdao 266021,China)

机构地区：[1]青岛大学转化医学研究院,山东青岛266021

出　　处：《精准医学杂志》2024年第4期289-294,共6页Journal of Precision Medicine

基　　金：国家自然科学基金面上项目(82070313)。

摘　　要：目的探究tRNA衍生的小RNA(tsRNA)对小鼠心肌细胞铁死亡的调控作用及其机制。方法采用实时荧光定量PCR(RT-qPCR)检测8周龄C57BL/6J小鼠心脏、肝脏、脾脏、肾脏、大脑、肌肉中5′tRF-LysCTT的相对表达水平。将原代心肌细胞分为A~D组,A组细胞使用完全培养基培养60 h,B组先使用完全培养基培养24 h,再依次进行饥饿处理12 h,H/R处理24 h,C组和D组细胞分别转染antagomir-NC和antagomir-5′tRF-LysCTT,并于转染24 h后再依次进行饥饿(12 h)和H/R处理(12 h);将原代心肌细胞分为E~G组,E组细胞使用完全培养基培养24 h,F组细胞转染agomir-NC 24 h,G组心肌细胞转染agomir-5′tRF-LysCTT 24 h。采用RT-qPCR检测A~G组小鼠心肌细胞中5′tRF-LysCTT和Ptgs2、Gpx4、Slc7a11的相对表达水平,分别采用亚铁离子比色法测试盒、脂质活性氧(ROS)染色、CCK8试剂盒检测A~G组小鼠心肌细胞内亚铁离子相对水平、ROS水平及心肌细胞的存活率,采用普鲁士蓝染色试剂盒观察A~G组小鼠心肌细胞内铁离子沉积情况。结果RT-qPCR检测结果显示,与肝脏、脾脏、肾脏、大脑、肌肉中相比较,小鼠心脏中5′tRF-LysCTT表达水平最高(F=16.21,t=3.81~7.93,P<0.05)。D组与B组相比,心肌细胞内5′tRF-LysCTT和Ptgs2相对表达水平降低、Gpx4以及Slc7a11相对表达水平升高、亚铁离子相对水平和ROS水平降低,心肌细胞的细胞存活率升高(t=3.26~15.61,P<0.05),细胞内铁离子沉积明显增多,而C组与B组相比,细胞内的上述指标的水平差异无显著性(P>0.05)。G组与E组相比,心肌细胞内5′tRF-LysCTT和Ptgs2相对表达水平升高、Gpx4和Slc7a11相对表达水平降低、亚铁离子相对水平和ROS水平升高,心肌细胞的细胞存活率降低(t=3.57~91.84,P<0.05),细胞内铁离子沉积明显减少,而F组与E组相比,细胞内的上述指标的水平比较差异无显著性(P>0.05)。结论5′tRF-LysCTT对于小鼠心肌细胞铁死亡具有调控作用,敲低细胞内5�Objective To investigate the regulatory effect of tRNA-derived small RNA(tsRNA)on ferroptosis in mouse cardiomyocytes and its mechanism.Methods RT-qPCR was used to measure the relative expression level of 5′tRF-LysCTT in the heart,liver,spleen,kidney,brain,and muscle of C57BL/6J mice aged 8 weeks.Primary cultured cardiomyocytes were divi-ded into groups A,B,C,and D:the cardiomyocytes in group A were cultured in a complete medium for 60 h;those in group B were cultured in a complete medium for 24 h,followed by starvation treatment for 12 h and H/R treatment for 24 h;those in groups C and D were transfected with antagomir-NC and antagomir-5′tRF-LysCTT,respectively,and were given starvation treatment(12 h)and H/R treatment(12 h)after 24 h of transfection.Primary cultured cardiomyocytes were divided into groups E,F,and G:the cardiomyocytes in group E were cultured in a complete medium for 24 h;those in group F were transfected with agomir-NC for 24 h;those in group G were transfected with agomir-5′tRF-LysCTT for 24 h.RT-qPCR was used to measure the relative expression levels of 5′tRF-LysCTT,Ptgs2,Gpx4,and Slc7a11 in mouse cardiomyocytes of groups A-G;ferrous ion colorimetric test kit,lipid reactive oxygen species(ROS)staining,and CCK8 assay kit were used to measure the relative content of ferrous ions,ROS level,and the survival rate of cardiomyocytes in groups A-G;Prussian blue staining was used to observe iron ion deposition in cardiomyocytes.Results RT-qPCR showed that the expression level of 5′tRF-LysCTT in heart was significantly higher than that in the liver,spleen,kidney,brain,and muscle(F=16.21,t=3.81-7.93,P<0.05).Compared with group B,group D had significant reductions in the relative expression levels of 5′tRF-LysCTT and Ptgs2,significant increases in the relative expression levels of Gpx4 and Slc7a11,significant reductions in the relative content of ferrous ions and ROS level,and a significant increase in the survival rate of cardiomyocytes(t=3.26-15.61,P<0.05),as well as a significant increase

关 键 词：RNA 转移 RNA 非翻译小片段 低氧 心肌再灌注损伤 肌细胞 心脏 铁死亡 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R542.2[医药卫生—基础医学]