[1]郭鸿浩,邓文文,龙禹哲,等.过表达LncRNA LOC100360491对缺氧复氧诱导H9c2心肌细胞凋亡的影响[J].第三军医大学学报,2020,42(18):1795-1802.
 GUO Honghao,DENG Wenwen,LONG Yuzhe,et al.LncRNA LOC100360491 overexpression inhibits hypoxiareoxygenationinduced apoptosis of H9c2 cells by upregulating PAK3 transcription[J].J Third Mil Med Univ,2020,42(18):1795-1802.
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第18期
页码:
1795-1802
栏目:
基础医学
出版日期:
2020-09-30

文章信息/Info

Title:
LncRNA LOC100360491 overexpression inhibits hypoxiareoxygenationinduced apoptosis of H9c2 cells by upregulating PAK3 transcription
作者:
郭鸿浩邓文文龙禹哲杨双亚赵永超石蓓
遵义医科大学附属医院心内科
Author(s):
GUO Honghao DENG Wenwen LONG Yuzhe YANG Shuangya ZHAO Yongchao SHI Bei

Department of Cardiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou Province, 563000, China

关键词:
长链非编码RNALncRNA LOC100360491心肌细胞缺氧复氧细胞凋亡
Keywords:
long noncoding RNA LancRNA LOC100360491 myocardial cells hypoxia reoxygenation apoptosis
分类号:
R322.11;R329.28;R394.2
文献标志码:
A
摘要:

目的观测长链非编码RNA(long noncoding RNA,LncRNA)LOC100360491(后简称LOC100360491)对缺氧复氧诱导的H9c2心肌细胞凋亡的影响,探讨其潜在作用机制。方法①通过慢病毒转染大鼠H9c2心肌细胞构建过表达及抑制LOC100360491的心肌细胞系,分为4组:空白对照组、过表达LOC100360491组、抑制LOC100360491组及空载体组,qRTPCR检测各组LOC100360491的表达。②将慢病毒转染的大鼠H9c2心肌细胞进行缺氧24 h复氧6 h处理以观察LOC100360491对该细胞凋亡的影响,分为5组:空白对照组、缺氧复氧组、过表达LOC100360491组、抑制LOC100360491组及空载体组。采用流式细胞术检测各组细胞凋亡早期磷脂酰丝氨酸外翻与凋亡中期线粒体活性氧释放情况;Western blot检测各组细胞中凋亡效应蛋白procaspase3、cleavedcaspase3、促凋亡蛋白Bax及抗凋亡蛋白Bcl2的表达。③采用qRTPCR检测各组PAK3及CNNM2基因的表达以探讨LOC100360491的潜在调控机制。结果①qRTPCR结果显示:与空载体组相比,过表达LOC100360491组LOC100360491表达显著上调(P<0.05),抑制LOC100360491组LOC100360491表达显著下调(P<0.05)。表明细胞系构建成功。②流式细胞术及Western blot结果显示,与缺氧复氧组相比,过表达LOC100360491组H9c2细胞早期凋亡、活性氧的释放均显著下降(P<0.05),cleavedcaspase3、Bax表达显著下调,Bcl2表达显著上调(P<0.05);而抑制LOC100360491组则与之相反,但差异无统计学意义。③qRTPCR结果显示:与空白对照组相比,过表达LOC100360491组PAK3表达显著上升(P<0.05),而CNNM2则无明显变化(P>0.05)。抑制LOC100360491组PAK3及CNNM2表达显著下降(P<0.05)。结论LOC100360491可通过线粒体活性氧途径抑制缺氧复氧诱导的大鼠H9c2心肌细胞凋亡的发生,可能与其在转录水平上调控PAK3的表达有关。
 

Abstract:

ObjectiveTo investigate the effect of  long noncoding RNA (LncRNA) LOC100360491 on apoptosis of H9c2 cardiomyocytes induced by hypoxiareoxygenation injury and explore the possible mechanism. MethodsH9c2 cells were infected with lentiviral vectors carrying LOC100360491 gene or its inhibitor (shLncRNA) or with a negative control vector, and the changes in LOC100360491 expression in the cells were detected by qRTPCR. The infected H9c2 cells were exposed to hypoxia for 24 h followed by reoxygenation for 6 h, and early apoptosis and reactive oxygen species (ROS) production of the cells were analyzed by flow cytometry. The expression of procaspase3, cleaved caspase3, Bax, and Bcl2 proteins in the cells were detected using Western blotting; the cellular expression levels of PAK3 and CNNM2 mRNA were detected using qRTPCR. ResultsThe results of qRTPCR confirmed successful overexpression or inhibition of LOC100360491 in H9c2 cells (P<0.05). Compared with the cells infected with the control lentiviral vector, the cells with lentivirusmediated LOC100360491 overexpression showed significantly decreased early apoptosis and ROS production with obviously downregulated expressions of cleaved caspase3 and Bax and upregulated Bcl2 expression following hypoxiareoxygenation (P<0.05). Inhibition of LOC100360491 in H9c2 cells resulted in the opposite changes, which were not significant compared with negative control group. Compared with the cells infected with the negative control vector, the cells with LOC100360491 suppression showed significantly downregulated expressions of PAK3 and CNNM2 mRNA (P<0.05), while LOC100360491 overexpression resulted in obviously upregulated PAK3 expression (P<0.05) without significantly affecting CNNM2 expression. ConclusionOverexpression of LOC100360491 inhibits apoptosis of H9c2 cells induced by hypoxiareoxygenation possibly by inhibiting mitochondrial release of ROS through upregulating the transcription of PAK3.

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更新日期/Last Update: 2020-09-22