circ_0023461 通过靶向 miR-370-3p/PDE4D 信号通路沉默保护心肌细胞免受缺氧诱导的功能障碍。
circ_0023461 Silencing Protects Cardiomyocytes from Hypoxia-Induced Dysfunction through Targeting miR-370-3p/PDE4D Signaling.
机构信息
Department of Cardiovascular Surgery, Xijing Hospital, Air Force Military Medical University, Xi'an, China.
Department of Cardiovascular Surgery, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China.
出版信息
Oxid Med Cell Longev. 2021 Oct 1;2021:8379962. doi: 10.1155/2021/8379962. eCollection 2021.
BACKGROUND
Acute myocardial infarction (AMI) is a common cardiovascular disease with high disability and mortality. Circular RNAs (circRNAs) are implicated in the pathomechanism of multiple human diseases, including AMI. This study intended to explore the function and working mechanism of a novel circRNA circ_0023461 in hypoxia-induced cardiomyocytes.
METHODS
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were implemented to detect RNA and protein expression. Cell counting kit-8 (CCK8) assay and 5-ethynyl-2'-deoxyuridine (Edu) assay were conducted to analyze cell viability and proliferation ability. Cell migration and apoptosis were assessed by Transwell assay and flow cytometry. Cell oxidative stress was analyzed using the commercial kits. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze cell inflammation. Cell glycolytic metabolism was evaluated using the commercial kits. Dual-luciferase reporter assay and RNA pull-down assay were conducted to verify the intermolecular interactions.
RESULTS
circ_0023461 expression was upregulated in AMI patients and hypoxia-induced AC16 cells. Hypoxia restrained the viability, proliferation, migration, and glycolysis and induced the apoptosis, oxidative stress, and inflammation of AC16 cells, and these effects were attenuated by the silence of circ_0023461. MicroRNA-370-3p (miR-370-3p) was verified as a target of circ_0023461, and circ_0023461 silencing-mediated protective effects in hypoxia-induced cardiomyocytes were partly alleviated by the knockdown of miR-370-3p. miR-370-3p interacted with the 3' untranslated region (3' UTR) of phosphodiesterase 4D (PDE4D), and PDE4D overexpression partly reversed miR-370-3p overexpression-induced protective effects in hypoxia-induced cardiomyocytes. circ_0023461 can upregulate PDE4D expression by acting as a molecular sponge for miR-370-3p in AC16 cells.
CONCLUSION
circ_0023461 knockdown attenuated hypoxia-induced dysfunction in AC16 cells partly by targeting the miR-370-3p/PDE4D axis.
背景
急性心肌梗死（AMI）是一种常见的心血管疾病，具有较高的残疾和死亡率。环状 RNA（circRNA）与包括 AMI 在内的多种人类疾病的发病机制有关。本研究旨在探讨新型环状 RNA circ_0023461 在缺氧诱导的心肌细胞中的功能和作用机制。
方法
采用逆转录定量聚合酶链反应（RT-qPCR）和 Western blot 检测 RNA 和蛋白质表达。通过细胞计数试剂盒-8（CCK8）和 5-乙炔基-2'-脱氧尿苷（Edu）检测分析细胞活力和增殖能力。通过 Transwell 检测和流式细胞术分析细胞迁移和凋亡。使用商业试剂盒分析细胞氧化应激。通过酶联免疫吸附测定（ELISA）分析细胞炎症。使用商业试剂盒评估细胞糖酵解代谢。通过双荧光素酶报告基因检测和 RNA 下拉实验验证分子间相互作用。
结果
AMI 患者和缺氧诱导的 AC16 细胞中 circ_0023461 的表达上调。缺氧抑制 AC16 细胞活力、增殖、迁移和糖酵解，并诱导细胞凋亡、氧化应激和炎症，而 circ_0023461 的沉默减轻了这些作用。微小 RNA-370-3p（miR-370-3p）被验证为 circ_0023461 的靶标，circ_0023461 沉默介导的缺氧诱导心肌细胞保护作用部分被 miR-370-3p 的敲低减轻。miR-370-3p 与磷酸二酯酶 4D（PDE4D）的 3'非翻译区（3'UTR）相互作用，PDE4D 的过表达部分逆转了 miR-370-3p 过表达诱导的缺氧诱导心肌细胞中的保护作用。circ_0023461 可以通过作为 AC16 细胞中 miR-370-3p 的分子海绵来上调 PDE4D 的表达。
结论
circ_0023461 的敲低部分通过靶向 miR-370-3p/PDE4D 轴减轻缺氧诱导的 AC16 细胞功能障碍。
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