circTLK1通过miR-26a-5p/YES1轴减轻小鼠心肌缺血/再灌注损伤  

circTLK1 attenuates myocardial ischemia/reperfusion injury in mice via the miR-26a-5p/YES1 axis

作　　者：蒋先训 张凯 张鹰 JIANG Xianxun;ZHANG Kai;ZHANG Ying(Department of Critical Care Medicine,the Second Affiliated Hospital,Hengyang Medical School,Univevsity of South China,Hengyang 421000,China)

机构地区：[1]南华大学衡阳医学院附属第二医院,重症医学科,湖南衡阳421000

出　　处：《中国比较医学杂志》2023年第8期86-94,共9页Chinese Journal of Comparative Medicine

基　　金：湖南省卫生健康委科研项目(202213014836);衡阳市科技计划项目(hyzdxjh202103);湖南省自然科学基金部门联合基金项目(2023JJ60356)。

摘　　要：目的探讨环状RNA凌乱样激酶1(circTLK1)在心肌缺血再灌注损伤(MIRI)中的作用和潜在机制。方法48只C57BL/6小鼠分为假手术组(Sham组)、MIRI组、sh-NC组、sh-circTLK1组、sh-circTLK1+antagomir-NC组、sh-circTLK1+antagomir-miR-26a-5p组,每组8只;采用结扎左冠状动脉前降支(LAD)建立MIRI小鼠模型。超声心动图检测小鼠左心室射血分数(EF)、左心室缩短分数(FS)、左心室舒张末期内径(LVEDD)和收缩末期内径(LVESD)以评估心脏功能,ELISA检测血清乳酸脱氢酶(LDH)、心肌肌钙蛋白I(cTnI)、肌酸激酶同工酶(CKMB)水平,苏木精-伊红(HE)染色检测心肌组织病理学变化,TUNEL染色检测心肌细胞凋亡,实时荧光定量PCR(qRT-PCR)和蛋白质印迹(Western blot)检测心肌组织中circTLK1、miR-26a-5p、YES1 mRNA和蛋白表达。双荧光素酶报告基因实验、RNA结合蛋白免疫沉淀(RIP)验证circTLK1/YES1和miR-26a-5p之间的相互作用。结果与Sham组相比,MIRI组小鼠EF、FS、心肌组织miR-26a-5p水平显著降低,LVEDD、LVESD、血清LDH、CK-MB活性和cTnI水平、心肌细胞凋亡率、心肌组织circTLK1、YES1 mRNA和蛋白水平显著升高(均P<0.05),心肌细胞排列紊乱,细胞间质有炎性细胞浸润;circTLK1敲低可显著上调miR-26a-5p,抑制YES1表达,改善上述指标变化(均P<0.05),减轻心肌损伤;抑制miR-26a-5p可通过上调YES1,显著减弱circTLK1敲低对MIRI的保护作用。双荧光素酶报告基因实验和RIP证实了circTLK1和miR-26a-5p之间的直接相互作用,YES1是miR-26a-5p的靶标。结论敲低circTLK1可能通过调节miR-26a-5p/YES1轴在MIRI中发挥保护作用,circTLK1可能是MIRI的潜在治疗靶点。Objective To investigate the role and underlying mechanism of circular RNA tousled-like kinase 1(circTLK1)in myocardial ischemia/reperfusion injury(MIRI).Methods Forty-eight C57BL/6 mice were divided into a sham operation group(Sham),MIRI group,sh-NC group,sh-circTLK1 group,sh-circTLK1+antagomir-NC group,and sh-circTLK1+antagomir-miR-26a-5p group with eight mice per group.The MIRI mouse model was established by ligation of the left anterior descending coronary artery.Left ventricular ejection fraction,left ventricular fractional shortening,left ventricular end-diastolic diameter,and end-systolic diameter were measured by echocardiography to assess cardiac functions.Serum levels of lactate dehydrogenase,cardiac troponin I and creatine kinase isoenzyme were measured by ELISA.Histopathological changes of myocardial tissue were assess by hematoxylin-eosin staining.Cardiomyocyte apoptosis was detected by TUNEL staining.mRNA and protein expression of circTLK1,miR-26a-5p,and YES1 in myocardial tissue were measured by real-time quantitative PCR and Western blot.An interaction between circTLK1/YES1 and miR-26a-5p was verified by dual luciferase reporter assays and RNA-binding protein immunoprecipitation.Results Compared with the findings in the Sham group,the ejection fraction,fractional shortening,and the level of myocardial tissue miR-26a-5p in the MIRI group were significantly decreased,the left ventricular end-diastolic diameter,end-systolic diameter,activities of serum lactate dehydrogenase and creatine kinase isoenzyme,the level of cardiac troponin I,myocardial cell apoptosis rate,and mRNA and protein levels of circTLK1 and YES1 in myocardial tissue were significantly increased(all P<0.05).Moreover,cardiomyocytes were disordered,and inflammatory cell infiltration was observed among interstitial cells.circTLK1 knockdown significantly upregulated miR-26a-5p expression,inhibited YES1 expression,improved the changes in the above indicators(all P<0.05),and reduced myocardial injury.Inhibition of miR-26a-5p significantly

关 键 词：心肌缺血再灌注损伤 环状RNA凌乱样激酶1 miR-26a-5p YES原癌基因1 细胞凋亡 

分 类 号：R-33[医药卫生]