MicroRNA-194-5p Attenuates Doxorubicin-Induced Cardiomyocyte Apoptosis and Endoplasmic Reticulum Stress by Targeting P21-Activated Kinase 2.
MicroRNA-194-5p Attenuates Doxorubicin-Induced Cardiomyocyte Apoptosis and Endoplasmic Reticulum Stress by Targeting P21-Activated Kinase 2.
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DOI：
10.3389/fcvm.2022.815916
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发表时间：
2022
影响因子：
3.6
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Fa H;Xiao D;Chang W;Ding L;Yang L;Wang Y;Wang M;Wang J
许多研究表明microRNAs（miRs）参与了阿霉素（DOX）心脏毒性的调控。据报道，miR-194- 5 p在心肌梗死患者中显著上调;然而，其在心肌疾病中的作用仍不清楚。各种刺激都可以触发内质网应激，最终激活细胞凋亡信号。本研究旨在探讨miR-194- 5 p在阿霉素诱导的内质网应激和心肌细胞凋亡中的调控作用。用2 μM DOX处理H9 c2以诱导细胞凋亡，这是刺激DOX诱导的心脏毒性模型。采用实时荧光定量PCR（qRT-PCR）检测miR-194- 5 p的表达，双荧光素酶报告基因分析检测miR-194- 5 p与P21激活激酶2（PAK 2）的相互作用，末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）法检测细胞凋亡，台盼蓝染色检测细胞死亡，流式细胞仪检测caspase-3/7活性。Western blotting检测蛋白表达;聚丙烯酰胺凝胶电泳和免疫荧光检测ER相关因子剪接X-box结合蛋白1（XBP 1 s），验证ER应激激活。在DOX处理的心肌细胞和小鼠心脏组织中，miR-194- 5 p上调，而PAK 2蛋白水平下调。PAK 2被预测为miR-194- 5 p的靶点;因此，双荧光素酶报告基因分析表明miR-194- 5 p直接与PAK 2相互作用并抑制其表达。TUNEL法、caspase-3/7活性检测和台盼蓝染色结果显示，抑制miR-194- 5 p或过表达PAK 2均减少DOX诱导的心肌细胞凋亡。miR-194- 5 p的沉默也改善了DOX诱导的心功能障碍。此外，DOX可诱导H9 c2的ER应激，从而导致XBP 1和caspase-12活化。XBP 1 s的表达水平在DOX处理后先升高后降低。XBP 1 s的过表达抑制了DOX诱导的caspase-3/7活性升高以及切割的caspase-12的表达，从而保护心肌细胞免于凋亡。此外，XBP 1 s的激活受miR-194- 5 p和PAK 2的调节。我们的研究结果表明，沉默miR-194- 5 p可以减轻DOX通过PAK 2和XBP 1 s在体外和体内诱导的心脏毒性。因此，新的miR-194- 5 p/PAK 2/XBP 1 s轴可能是接受DOX治疗的癌症患者的潜在预防/治疗靶点。
Many studies have reported that microRNAs (miRs) are involved in the regulation of doxorubicin (DOX)-induced cardiotoxicity. MiR-194-5p has been reported significantly upregulated in patients with myocardial infarction; however, its role in myocardial diseases is still unclear. Various stimuluses can trigger the endoplasmic reticulum (ER) stress and it may activate the apoptosis signals eventually. This study aims to explore the regulatory role of miR-194-5p in DOX-induced ER stress and cardiomyocyte apoptosis. H9c2 was treated with 2 μM DOX to induce apoptosis, which is to stimulate the DOX-induced cardiotoxicity model. The expression of miR-194-5p was detected by quantitative real-time PCR (qRT-PCR); the interaction between miR-194-5p and P21-activated kinase 2 (PAK2) was tested by dual luciferase reporter assay; terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and caspase-3/7 activity were used to assess apoptosis; trypan blue staining was applied to measure cell death; Western blotting was performed to detect protein expressions; and ER-related factors splicing X-box binding protein 1 (XBP1s) was detected by polyacrylamide gel electrophoresis and immunofluorescence to verify the activation of ER stress. MiR-194-5p was upregulated in cardiomyocytes and mouse heart tissue with DOX treatment, while the protein level of PAK2 was downregulated. PAK2 was predicted as the target of miR-194-5p; hence, dual luciferase reporter assay indicated that miR-194-5p directly interacted with PAK2 and inhibited its expression. TUNEL assay, caspase-3/7 activity test, and trypan blue stain results showed that either inhibition of miR-194-5p or overexpression of PAK2 reduced DOX-induced cardiomyocyte apoptosis. Silencing of miR-194-5p also improved DOX-induced cardiac dysfunction. In addition, DOX could induce ER stress in H9c2, which led to XBP1 and caspase-12 activation. The expression level of XBP1s with DOX treatment increased first then decreased. Overexpression of XBP1s suppressed DOX-induced caspase-3/7 activity elevation as well as the expression of cleaved caspase-12, which protected cardiomyocyte from apoptosis. Additionally, the activation of XBP1s was regulated by miR-194-5p and PAK2. Our findings revealed that silencing miR-194-5p could alleviate DOX-induced cardiotoxicity via PAK2 and XBP1s in vitro and in vivo. Thus, the novel miR-194-5p/PAK2/XBP1s axis might be the potential prevention/treatment targets for cancer patients receiving DOX treatment.