病毒性心肌炎血清外泌体来源的miR-320通过靶向Pik3r1抑制AKT/mTOR通路促进小鼠心肌细胞凋亡  

Viral myocarditis serum exosome-derived miR-3200 promotes the apoptosis of mouse cardiomyocytesSby inhibiting AKT/mTOR pathway and targeting phosphatidylinositol 3-kinase regulatory subunit 1(Pik3r1)

作　　者：张欣 李雪琴[1,2] 朱良宇[1,2] 殷国泉[1,2] 张苑 吕坤 ZHANG Xin;LI Xueqin;ZHU Liangyu;YIN Cuoquan;ZHANG Yuan;LYU Kun(Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institution;Central Laboratory,Yijishan Hospital,Wannan Medical College,Wuhu 241001,China)

机构地区：[1]皖南医学院重大疾病非编码RNA转化研究安徽普通高校重点实验室 [2]皖南医学院弋矶山医院中心实验室,安徽芜湖241001

出　　处：《细胞与分子免疫学杂志》2023年第6期516-525,共10页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(82072370)。

摘　　要：目的探讨病毒性心肌炎血清外泌体miR-320对心肌细胞凋亡的影响及机制。方法使用柯萨奇病毒B3(CVB3)腹腔注射,建立病毒性心肌炎小鼠模型;采用血清外泌体抽提试剂盒提取血清外泌体,与心肌细胞共培养,激光共聚焦显微镜观察心肌细胞摄取外泌体情况;使用miR-320抑制剂或模拟物转染心肌细胞,实时荧光定量PCR检测miR-320表达水平;采用流式细胞术检测心肌细胞凋亡率,Western blot法检测B细胞淋巴瘤因子2(Bcl2)和Bcl2相关X蛋白(BAX)表达水平;生物信息学预测miR-320靶基因并进行基因本体论(GO)和京都基因和基因组百科全书(KEGG)富集分析;荧光素酶报告基因检测miR-320与其靶基因磷脂酰肌醇3激酶调节亚基1(Pik3r1)的作用关系;Western blot法检测miR-320对蛋白激酶B/哺乳动物雷帕霉素靶蛋白(AKT/mTOR)通路蛋白的影响。结果病毒性心肌炎血清外泌体促进心肌细胞凋亡并上调BAX水平,同时降低Bcl2水平;病毒性心肌炎小鼠心肌组织中miR-320显著上调,且miR-320成熟体和miR-320前体pri-miR-320表达在心肌细胞均明显上调;病毒性心肌炎血清外泌体处理的心肌细胞中miR-320水平显著上调,而转染miR-320抑制剂逆转了外泌体引起的miR-320上调及凋亡率升高;Pik3r1是miR-320的靶基因,其过表达逆转miR-320上调导致的心肌细胞凋亡;miR-320过表达抑制AKT/mTOR通路激活。结论病毒性心肌炎血清外泌体miR-320通过靶向Pik3r1抑制AKT/mTOR通路促进小鼠心肌细胞凋亡。Objective To investigate the effect of viral myocarditis serum exosomal miR-320 on apoptosis of cardiomyocytes and its mechanism.Methods The model of viral myocarditis mice was established by intraperitoneal injection of Coxsackie virus B3.Serum exosomes were extracted by serum exosome extraction kit and co-cultured with cardiomyocytes.The uptake of exosomes by cardiomyocytes was detected by laser confocal microscopy.Cardiomyocytes were transfected with miR-320 inhibitor or mimic,and the expression level of miR-320 was detected by real-time quantitative PCR.Flow cytometry was used to detect cardiomyocyte apoptosis rate,and the expression levels of B cell lymphoma 2(Bcl2)and Bcl2-related X protein(BAX)were tested by Western blot analysis.The prediction of miR-320 target genes and GO and KEGG enrichment analysis were tested by online database.The relationship between miR-320 and its target gene phosphoinositide-3-kinase regulatory subunit 1(Pik3r1)was examined by luciferase reporter gene.The effect of miR-320 on AKT/mTOR pathway protein was detected by Western blot analysis.Results Viral myocarditis serum exosomes promoted cardiomyocyte apoptosis,and increased the level of BAX while the level of Bcl2 was decreased.miR-320 was significantly up-regulated in myocardial tissue of viral myocarditis mice,and both pri-miR-320 and mature of miR-320 were up-regulated greatly in cardiomyocytes.The level of miR-320 in cardiomyocytes treated with viral myocarditis serum exosomes was significantly up-regulated,while transfection of miR-320 inhibitor counteracted miR-320 overexpression and reduced apoptosis rate caused by exosomes.Pik3rl is the target gene of miR-320,and its overexpression reversed cardiomyocyte apoptosis induced by miR-320 up-regulation.The overexpression of miR-320 inhibited AKT/mTOR pathway activation.Conclusion Viral myocarditis serum exosome-derived miR-320 promotes apoptosis of mouse cardiomyocytes by inhibiting AKT/mTOR pathway by targeting Pik3rl.

关 键 词：病毒性心肌炎 外泌体 磷脂酰肌醇3激酶调节亚基1(Pik3r1) miR-320 心肌细胞 凋亡 

分 类 号：R392-33[医药卫生—免疫学] R542.21[医药卫生—基础医学] Q291[生物学—细胞生物学]