[1]黄 毅,王琮翰.miR-330-5p调控Nox4对缺氧复氧大鼠胚胎心肌细胞损伤影响的实验研究[J].现代检验医学杂志,2024,39(06):48-53+83.[doi:10.3969/j.issn.1671-7414.2024.06.008]
　HUANG Yi,WANG Conghan.Experimental Study on the Effect of miR-330-5p Regulated Nox4 on the Damage of Fetal Cardiomyocytes in Hypoxic-reoxygenated Rats[J].Journal of Modern Laboratory Medicine,2024,39(06):48-53+83.[doi:10.3969/j.issn.1671-7414.2024.06.008]
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miR-330-5p调控Nox4对缺氧复氧大鼠胚胎心肌细胞损伤影响的实验研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年06期
页码:
48-53+83
栏目:
论著
出版日期:
2024-11-15

文章信息/Info

Title:
Experimental Study on the Effect of miR-330-5p Regulated Nox4 on the Damage of Fetal Cardiomyocytes in Hypoxic-reoxygenated Rats
文章编号:
1671-7414（2024）06-048-07
作者:
黄 毅; 王琮翰
（西安市人民医院心血管内科，西安 710004）
Author(s):
HUANG Yi;  WANG Conghan
（Department of Cardiovascular Medicine, Xi’an People’s Hospital, Xi’an 710004, China）
关键词:
微小RNA-330-5p; 烟酰胺腺嘌呤二核苷酸磷酸氧化酶4; 缺氧/ 复氧; 心肌细胞; 凋亡
分类号:
R-332
DOI:
10.3969/j.issn.1671-7414.2024.06.008
文献标志码:
A
摘要:
目的 研究miR-330-5p 调控烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（nicotinamide adenine dinucleotide phosphateoxidase 4，Nox4）对缺氧/ 复氧（hypoxia/reoxygenation，H/R）大鼠胚胎心肌细胞损伤的影响及可能机制。方法 将大鼠胚胎心肌细胞H9C2 分为对照组（Con）、模型组（H/R）、H/R+anti-miR-NC 组、H/R+anti-miR-330-5p 组、H/R+pcDNA3.1 组、H/R+pcDNA3.1-Nox4 组、H/R+anti-miR-330-5p+si-NC 组和H/R+anti-miR-330-5p+si-Nox4 组。实时荧光定量聚合酶链式反应（qRT-PCR）检测miR-330-5p 在H/R 诱导心肌细胞中的表达；MTT 法和流式细胞术检测心肌细胞增殖活力和细胞凋亡；Western blot 检测Nox4 蛋白及凋亡相关蛋白[ 细胞周期素D1（Cyclin D1）、B 淋巴细胞瘤-2（B cell lymphoma/leukemia-2，Bcl-2）、Bcl-2 相关X 蛋白（Bcl-2 associated X，Bax）、P21] 表达；双荧光素酶报告基因实验验证miR-330-5p 和Nox4 的靶向关系。结果 与Con 组比较，H/R 组细胞中miR-330-5p 表达（1.96±0.20 vs1.01±0.11），P21（0.89±0.07 vs 0.21±0.03）和Bax（0.80±0.05 vs 0.18±0.02）蛋白水平及细胞凋亡率（26.67%±2.68%vs 7.22%±0.68%）明显升高（t=7.029，12.591，12.790，12.102），而Cyclin D1（0.20±0.03 vs 0.78±0.06）和Bcl-2（0.11±0.02vs 0.71±0.06）蛋白水平及细胞增殖活性显著降低（t=13.671；11.047；11.333，11.485，12.511），差异具有统计学意义（均P <0.01）。Nox4 是miR-330-5p 的靶基因，miR-330-5p 负调控Nox4 表达。抑制miR-330-5p 表达或过表达Nox4 可促进Cyclin D1 和Bcl-2 蛋白表达（t=9.664，7.548），抑制P21 和Bax 蛋白表达（t=10.184，10.314），增强心肌细胞增殖活力（t=6.667~9.126）、抑制细胞凋亡（t=10.341），差异具有统计学意义（均P<0.01）。抑制Nox4 表达能够逆转抑制miR-330-5p 对H/R 心肌细胞增殖（t=4.146~7.941）和凋亡（t=6.285~11.358）的影响（均P<0.01）。结论 H/R 诱导的大鼠心肌细胞中miR-330-5p 表达上调，抑制miR-330-5p 可能通过靶向Nox4 抑制心肌细胞凋亡，对H/R 大鼠心肌细胞损伤起到保护作用。
Abstract:
Objective To study the effect of miR-330-5p on nicotinamide adenine dinucleotide phosphate oxidase 4（Nox4） on hypoxia/reoxygenation (H/R) effect and possible mechanism of myocardial cell injury in rat embryo. Methods Rat embryonic cardiomyocytes H9C2 were divided into control group (Con), model group (H/R), H/R+anti-miR-NC group, H/ R+anti-miR-330-5p group, H/R+pcDNA3.1 group, H/R+ PCDNA3.1-NOX4 group, and H/R+anti-miR-330-5p+si -NC group and H/R+anti-miR-330-5p+si-Nox4 group. Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-330-5p in H/ R-induced cardiomyocytes. The proliferation and apoptosis of cardiomyocytes were detected by MTT and flow cytometry. Western blot was used to detect Nox4 protein and apoptosis-related protein [Cyclin D1 (Cyclin D1), B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 Associated X (Bcl-2 Associated X), P21] expression, and the dual luciferase reporter gene assay verified the targeting relationship between miR-330-5p and Nox4. Results Compared with Con group, the expression of miR-330-5p(1.96±0.20 vs 1.01±0.11), P21(0.89±0.07 vs 0.21±0.03)and Bax(0.80±0.05 vs 0.18±0.02) protein levels and cell apoptosis rate(26.67%±2.68% vs 7.22%±0.68%) in H/R group were increased (t=7.029, 12.591, 12.790, 12.102), while Cyclin D1(0.20±0.03 vs 0.78±0.06 ) and Bcl-2(0.11±0.02 vs 0.71±0.06 ) protein levels and cell proliferation activity were decreased（t=13.671; 11.047; 11.333, 11.485, 12.511），the differences were statistically significant (all P<0.01). Nox4 was a target gene of miR-330-5p, and miR-330-5p negatively regulates Nox4 expression. Inhibition of miR-330-5p expression or overexpression of Nox4 can promote the expression of Cyclin D1 and Bcl-2 proteins（t=9.664，7.548）, inhibit the expression of P21 and Bax proteins, enhance the proliferation activity of cardiomyocytes（t=10.184, 10.314）, enhance the proliferation activity of myocardial cells (t=6.667~9.126) and inhibit cell apoptosis（t=10.341）, and the differences were statistically significant (all P<0.01). Inhibition of Nox4 expression can reverse the effects of miR-330-5p on proliferation(t=4.146~7.941, all P<0.05) and apoptosis of H/R cardiomyocytes (t=6.285~11.358, all P<0.01). Conclusion H/ R-induced expression of miR-330- 5p is up-regulated in rat cardiomyocytes. Inhibition of miR-330-5p may inhibit cardiomyocyte apoptosis through targeting Nox4, and play a protective role in myocardial cell damage in H/R rats.

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备注/Memo

备注/Memo:
作者简介：黄毅(1984-)，男，本科，主治医师，研究方向：冠心病、心律失常、高血压，E-mail：huangyiyx@163.com。
通讯作者：王琮翰(1989-)，男，硕士研究生，中级职称，研究方向：心血管内科相关疾病，E-mail：511856036@qq.com。
更新日期/Last Update: 2024-11-15