LncRNA LOC100360491 overexpression inhibits hypoxia-reoxygenation-induced apoptosis of H9c2 cells by upregulating PAK3 transcription

Honghao GUO; Wenwen DENG; Yuzhe LONG; Shuangya YANG; Yongchao ZHAO; Bei SHI

doi:10.16016/j.1000-5404.202004065

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LncRNA LOC100360491 overexpression inhibits hypoxia-reoxygenation-induced apoptosis of H9c2 cells by upregulating PAK3 transcription

Basic Medicine | 更新时间：2026-01-09

- LncRNA LOC100360491 overexpression inhibits hypoxia-reoxygenation-induced apoptosis of H9c2 cells by upregulating PAK3 transcription
- Journal of Third Military Medical University Vol. 42, Issue 18, Pages: 1795-1802(2020)
- 作者机构：
  
  563000 贵州遵义，遵义医科大学附属医院心内科
- 作者简介：
- 基金信息：
  
  the Regional Science Research Program of National Natural Science Foundation of China(81560041)
- DOI：10.16016/j.1000-5404.202004065
  CLC： R322.11;R329.28;R394.2
- Received：07 April 2020，
  
  Revised：2020-06-22，
  
  Published：30 September 2020
- 稿件说明：
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Honghao GUO, Wenwen DENG, Yuzhe LONG, et al. LncRNA LOC100360491 overexpression inhibits hypoxia-reoxygenation-induced apoptosis of H9c2 cells by upregulating PAK3 transcription[J]. Journal of Third Military Medical University, 2020, 42(18): 1795-1802.
DOI：

Honghao GUO, Wenwen DENG, Yuzhe LONG, et al. LncRNA LOC100360491 overexpression inhibits hypoxia-reoxygenation-induced apoptosis of H9c2 cells by upregulating PAK3 transcription[J]. Journal of Third Military Medical University, 2020, 42(18): 1795-1802. DOI： 10.16016/j.1000-5404.202004065.

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

① qRT-PCR结果显示:与空载体组相比，过表达LOC100360491组LOC100360491表达显著上调(

0.05)，抑制LOC100360491组LOC100360491表达显著下调(

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(

0.05)，cleaved-caspase3、Bax表达显著下调，Bcl-2表达显著上调(

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(

0.05)，而CNNM2则无明显变化(

0.05)。抑制LOC100360491组PAK3及CNNM2表达显著下降(

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

Objective

To investigate the effect of long noncoding RNA (LncRNA) LOC100360491 on apoptosis of H9c2 cardiomyocytes induced by hypoxia-reoxygenation injury and explore the possible mechanism.

Methods

H9c2 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.

Results

The results of qRT-PCR confirmed successful overexpression or inhibition of LOC100360491 in H9c2 cells (

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 (

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 (

0.05)

while LOC100360491 overexpression resulted in obviously up-regulated PAK3 expression (

0.05) without significantly affecting CNNM2 expression.

Conclusion

Overexpression of LOC100360491 inhibits apoptosis of H9c2 cells induced by hypoxia-reoxygenation possibly by inhibiting mitochondrial release of ROS through upregulating the transcription of PAK3.

关键词

Keywords

references

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