Chen Baozeng, Zheng Lingling, Zhu Teng, Jiao Kai
Department of Cardiology, The second people's hospital of Liaocheng, Liaocheng, Shandong, China.
Department of Cardiovascular Medicine, Shengli Oilfield Central Hospital, Dongying, Shandong, China.
J Biochem Mol Toxicol. 2022 Dec;36(12):e23218. doi: 10.1002/jbt.23218. Epub 2022 Sep 13.
Long noncoding RNA forkhead box D3-antisense RNA 1 (FOXD3-AS1) is associated with cardiovascular diseases, but its roles in myocardial ischemia/reperfusion (I/R) injury and the related signaling pathway have not been fully reported. We aimed to investigate the roles and mechanism of action of FOXD3-AS1 in myocardial I/R injury. An in vivo myocardial I/R injury mouse model and an in vitro hypoxia/reoxygenation (H/R) cardiomyocyte model was established. Quantitative reverse transcription-polymerase chain reaction, western blotting, and immunofluorescent assays were performed to examine the expression levels of FOXD3-AS1, microRNA (miR)-128, thioredoxin-interacting protein/regulation of development and DNA damage response 1/protein kinase B/glycogen synthase kinase 3β/nuclear factor erythroid 2-related factor 2 (TXNIP/Redd1/AKT/GSK3β/Nrf2) pathway-related proteins and apoptosis-related proteins. The interactions between FOXD3-AS1 and miR-128 and miR-128 and TXNIP were analyzed by Spearman's correlation test, predicted by ENCORI, and verified by dual-luciferase reporter assay. In addition, the levels of cardiac injury markers and oxidative stress markers were evaluated by corresponding kits. Cell Counting Kit-8 assays and flow cytometry were performed to assess cell viability and apoptosis. Hematoxylin and eosin staining was applied to observe the effect of FOXD3-AS1 on the morphology of myocardial I/R injured tissues. The results showed that the FOXD3-AS1 and TXNIP were highly expressed, whereas miR-128 was expressed at low levels in I/R myocardial tissues and H/R-induced H9c2 cells. FOXD3-AS1 directly targeted miR-128 to reduce its expression. TXNIP was confirmed as a downstream target of miR-128. Knockdown of FOXD3-AS1 led to the alleviation of I/R injury in vivo and in vitro. FOXD3-AS1 enhanced the expression of TXNIP by sponging miR-128, which inhibited the Redd1/AKT/GSK3β/Nrf2 pathway. Both inhibition of miR-128 and overexpression of TXNIP reversed the cardioprotective effect of FOXD3-AS1 small interfering RNA in H/R-induced H9c2 cells.
长链非编码RNA叉头框D3反义RNA1（FOXD3-AS1）与心血管疾病有关，但其在心肌缺血/再灌注（I/R）损伤及相关信号通路中的作用尚未完全报道。我们旨在研究FOXD3-AS1在心肌I/R损伤中的作用及作用机制。建立了体内心肌I/R损伤小鼠模型和体外缺氧/复氧（H/R）心肌细胞模型。采用定量逆转录-聚合酶链反应、蛋白质印迹法和免疫荧光分析检测FOXD3-AS1、微小RNA（miR）-128、硫氧还蛋白相互作用蛋白/发育调控和DNA损伤反应1/蛋白激酶B/糖原合酶激酶3β/核因子红细胞2相关因子2（TXNIP/Redd1/AKT/GSK3β/Nrf2）通路相关蛋白及凋亡相关蛋白的表达水平。通过Spearman相关性检验分析FOXD3-AS1与miR-128以及miR-128与TXNIP之间的相互作用，由ENCORI进行预测，并通过双荧光素酶报告基因检测进行验证。此外，使用相应试剂盒评估心脏损伤标志物和氧化应激标志物的水平。采用细胞计数试剂盒-8检测和流式细胞术评估细胞活力和凋亡情况。应用苏木精-伊红染色观察FOXD3-AS1对心肌I/R损伤组织形态的影响。结果显示，在I/R心肌组织和H/R诱导的H9c2细胞中，FOXD3-AS1和TXNIP高表达，而miR-128低表达。FOXD3-AS1直接靶向miR-128以降低其表达。TXNIP被证实为miR-128的下游靶点。敲低FOXD3-AS1可减轻体内和体外的I/R损伤。FOXD3-AS1通过吸附miR-128增强TXNIP的表达，从而抑制Redd1/AKT/GSK3β/Nrf2通路。抑制miR-128和过表达TXNIP均可逆转FOXD3-AS1小干扰RNA对H/R诱导的H9c2细胞的心脏保护作用。
