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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (5): 53-62    DOI: 10.13523/j.cb.20190506
研究报告     
miR-186-5p在酒精诱导的心肌细胞中高表达并通过靶基因XIAP调控细胞凋亡水平 *
刘叶(),潘玥,郑魏,胡晶
辽宁省锦州医科大学附属第一医院心电中心 锦州 121000
miR-186-5p is Expressed Highly in Ethanol-induced Cardiomyocytes and Regulates Apoptosis by Target Gene XIAP
Ye LIU(),Yue PAN,Wei ZHENG,Jing HU
Electrocardial Center of the First Affiliated Hospital of Jinzhou Medical University,Jinzhou 121000, China
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摘要：

目的:证实酒精可诱导AC16心肌细胞凋亡及其与酒精浓度和作用时间的关系,研究不同浓度酒精干预下AC16心肌细胞中miR-186-5p与X连锁凋亡抑制蛋白(XIAP)表达水平以及心肌细胞凋亡水平的改变,探究miR-186-5p以XIAP为靶基因调控酒精诱导的心肌细胞凋亡。方法:流式细胞术检测细胞凋亡水平,Western blot、实时定量PCR技术分别在蛋白质及基因水平检测细胞miR-186-5p与XIAP表达水平的变化,双萤光素酶报告基因靶基因荧光检测miR-186-5p与XIAP的靶际关系。结果:酒精诱导AC16心肌细胞发生凋亡,且与酒精浓度及作用时间呈正相关;酒精摄入上调AC16心肌细胞中miR-186-5p表达,下调XIAP表达;miR-186-5p参与酒精诱导的AC16心肌细胞凋亡过程,XIAP抑制酒精诱导的AC16心肌细胞凋亡;miR-186-5p以XIAP为靶基因调控酒精诱导的心肌细胞凋亡。结论:AC16心肌细胞经过酒精处理后,细胞的凋亡水平升高,并且随着酒精作用浓度和作用时间的延长,凋亡水平进一步升高;酒精处理后心肌细胞中miR-186-5p表达量上调,XIAP表达量下调,miR-186-5p以XIAP为靶基因,调控酒精处理后心肌细胞的凋亡。
关键词： 心肌细胞;  细胞凋亡;  miR-186-5p;  X连锁凋亡抑制蛋白;  酒精性心肌病    
Abstract:

Objective: To confirm the effect of alcohol-induced apoptosis of AC16 cardiomyocytes and the relationship with the alcohol concentration and the action time.To investigate the expression levels of miR-186-5p and X-linked inhibitor of apoptosis protein(XIAP) along with the variational apoptosis level in alcohol -treated AC16 cardiomyocytes with different concentrations. To explore miR-186-5p regulated apoptosis of alcohol-induced cardiomyocytes using XIAP as the target gene.Methods:Flow cytometry analyzed the expression levels of cardiomyocyte apoptosis. Western blot and real-time PCR detected respectly the protein and RNA expression levels of miR-186-5p and XIAP in alcohol-treated AC16 cardiomyocytes. Luciferase reporter gene experiment verified that XIAP is a direct target gene of miR-186-5p.Results:Alcohol induced cardiomyocyte apoptosis, and the levels of apoptosis were dependent on alcohol concentration and duration of time. Alcohol intake can increase the expression levels of miR-186-5p and decrease the expression levels of XIAP in AC16 cardiomyocytes. miR-186-5p was involved in alcohol-induced the process of AC16 cardiomyocyte apoptosis.XIAP can inhibite alcohol-induced AC16 cardiomyocyte apoptosis.miR-186-5p targets XIAP as a target gene to regulate alcohol-induced cardiomyocyte apoptosis.Conclusion:The levels of apoptosis in AC16 cardiomyocytes increase after alcohol treatment and increase further with the increase of alcohol concentration and the prolongation of the action time. The expression levels of miR-186-5p increase, and the expression levels of XIAP decrease in alcohol-treated AC16 cardiomyocytes. miR-186-5p regulates apoptosis of alcohol-induced cardiomyocytes using XIAP as the target gene.
Key words: Cardiomyocyte    Apoptosis    miR-186-5p    XIAP    Alcoholic cardiomyopathy
收稿日期: 2018-10-24 出版日期: 2019-06-04
ZTFLH:  R542.2  
基金资助: * 辽宁省自然科学基金指导计划资助项目(20180550419)
通讯作者: 刘叶     E-mail: 601924547@qq.com
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引用本文:

刘叶,潘玥,郑魏,胡晶. miR-186-5p在酒精诱导的心肌细胞中高表达并通过靶基因XIAP调控细胞凋亡水平 *[J]. 中国生物工程杂志, 2019, 39(5): 53-62.

Ye LIU,Yue PAN,Wei ZHENG,Jing HU. miR-186-5p is Expressed Highly in Ethanol-induced Cardiomyocytes and Regulates Apoptosis by Target Gene XIAP. China Biotechnology, 2019, 39(5): 53-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190506    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I5/53

共转染 miR-186-5p mimic control pmir GLO-wt-XIAP-3'-UTR
共转染 miR-186-5p mimic pmir GLO-wt-XIAP-3'-UTR
共转染 miR-186-5p mimic control pmir GLO-mut-XIAP-3'-UTR
共转染 miR-186-5p mimic pmir GLO-mut-XIAP-3'-UTR
  
图1  流式细胞术分析酒精处理后的AC16心肌细胞的凋亡水平
图2  萤光素酶报告基因验证XIAP是miR-186-5p的直接作用靶点
图3  实时荧光定量PCR检测酒精处理后AC16心肌细胞中miR-186-5p的表达水平
图4  Western blot和实时荧光定量PCR检测酒精摄入后AC16心肌细胞中XIAP的表达水平
图5  流式细胞术分析细胞转染miR-186-5p mimic对酒精诱导的心肌细胞凋亡水平的影响
图6  流式细胞术分析细胞转染XIAP质粒对酒精诱导的心肌细胞凋亡水平的影响
图7  Western blot和实时荧光定量PCR检测转染miR-186-5p mimic/inhibitor后AC16心肌细胞中XIAP表达水平
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