Isoflurane alleviates hypoxia/reoxygenation induced myocardial injury by reducing miR-744 mediated SIRT6
Isoflurane alleviates hypoxia/reoxygenation induced myocardial injury by reducing miR-744 mediated SIRT6
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DOI：
10.1080/15376516.2021.1995556
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发表时间：
2021-11-09
影响因子：
3.2
通讯作者：
Feng, Zeguo
中科院分区：
文献类型：
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作者：
Chen, Guoqing;Zhang, Faqiang;Feng, Zeguo
本研究的目的是探讨miR-744及其靶基因在ISO对缺氧/复氧（H/R）诱导的心肌损伤的保护中的作用。方法采用体外培养的大鼠心肌细胞H9c2建立H/R模型，采用荧光定量PCR检测miR-744 mRNA的表达水平。CCK-8和流式细胞仪检测细胞活力和凋亡。采用酶联免疫吸附试验（ELISA）检测心肌损伤标志物CK-MB、cTnI和LDH。在线生物信息学软件miRDB和miRWalk预测了miR-744的靶点及其潜在结合位点，并通过荧光素酶报告基因分析验证了该靶点。结果H/R诱导后，miR-744 mRNA水平明显升高，细胞活力下降，凋亡增加（p <0.05）。心肌损伤标志物CK-MB、cTnI和LDH表达也增加（p <0.05）。而ISO预处理可明显减轻H/R引起的细胞存活率下降、细胞凋亡增加及心肌损伤标志物的升高，起到心肌保护作用（p <0.05）。更重要的是，miR-744的升高明显削弱了ISO对H/R诱导的心肌损伤的保护作用，导致细胞存活率降低，凋亡增加，心肌损伤指标浓度升高（p <0.05）。荧光素酶报告基因分析证实，Sirtuins6（SIRT6）是miR-744的潜在靶点，在H/R诱导的心肌损伤中降低，ISO暴露可逆转其水平（p <0.05）。结论ISO预处理可显著调节miR-744及其下游靶点SIRT6的表达，减轻H/R所致的心肌损伤。
Background The objective of this study was to investigate the role of miR-744 and its target genes in ISO protection against hypoxia/reoxygenation (H/R) induced myocardial injury. Methods Rat cardiomyocytes H9c2 was used to establish an H/R model in vitro, and the level of miR-744 mRNA was detected by fluorescence quantitative PCR. CCK-8 and flow cytometry was used to detected cell viability and apoptosis. Myocardial injury markers CK-MB, cTnI, and LDH were detected by enzyme-linked immunosorbent assay (ELISA). Online bioinformatics software miRDB and miRWalk predicts miR-744 target and its potential binding site, and verifies the target by luciferase reporter assay. Results After H/R induction, miR-744 mRNA level was remarkedly increased, cell viability was deceased, and apoptosis was increased (p < 0.05). Myocardial injury markers CK-MB, cTnI, and LDH expressions were also increased (p < 0.05). However, ISO pretreatment can significantly alleviate the decrease in cell viability induced by H/R, the increase of cell apoptosis, and the increase of myocardial injury markers, and it play a cardioprotective effect (p < 0.05). More importantly, elevated miR-744 remarkedly weakened the protective effect of ISO on H/R-induced myocardial injury, resulting in decreased cell viability, increased apoptosis, and elevated concentration of myocardial injury indicators (p < 0.05). Luciferase reporter assay confirmed that Sirtuins6 (SIRT6) is a potential target of miR-744 and decreased in H/R-induced myocardial injury, and ISO exposure can reverse its level (p < 0.05). Conclusion Our findings provide new insights that ISO pretreatment can remarkedly regulate miR-744 and its downstream target SIRT6 to mitigate myocardial injury induced by H/R.