Lou Yunpeng, Wang Shiying, Qu Jinlong, Zheng Jinhao, Jiang Weiwei, Lin Zhaofen, Zhang Sheng
Department of Emergency and Critical Care, Changzheng Hospital, Second Military Medical University Shanghai, China.
Department of Critical Care Medicine, 401 Hospital of PLA Qingdao, Shandong, China.
Int J Clin Exp Pathol. 2018 Jul 1;11(7):3222-3235. eCollection 2018.
As a complex pathophysiological event, myocardial ischemia/reperfusion injury (IRI) can cause heart failure, which has been associated with pyroptosis, a pro-inflammatory programmed cell death. Small endogenous non-coding RNAs have been shown to be involved in myocardial IRI. In the present study, we aimed to investigate whether miR-424 modulated pyroptosis in response to myocardial IRI and determine its underlying regulatory mechanism. An mouse model of cardiac IRI was established, and contractile function was evaluated by echography. The serum and heart tissue were harvested 24 h after reperfusion to assess the status of pyroptosis. For the study, H9C2 cells (a rat heart cell line) were subjected to 6 h of hypoxia, followed by 18 h of reoxygenation. The gene expressions at the mRNA level were assessed by real-time PCR, and the expressions at the protein level were examined by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). Bioinformatic analysis was applied to predict miR-424 targets, which were then confirmed by a luciferase reporter assay. We found that the expressions of pyroptosis-related proteins, including caspase-1, caspase-11, IL-1β, and IL-18, were significantly increased upon myocardial IRI. Similarly, hypoxia/reoxygenation injury (HRI) also induced pyroptosis in H9C2 cells. Furthermore, our study revealed that the miR-424 expression was substantially increased in I/R heart tissue and H/R-challenged H9C2 cells. In addition, we found that exogenous expression of miR-424 directly targeted cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and up-regulated the expressions of caspase-1 and the pro-inflammatory cytokines IL-1β and IL-18. Taken together, our findings provided a new signaling pathway of miR-424/CRISPLD2 in cardiac pyroptosis under IRI conditions.
作为一种复杂的病理生理事件，心肌缺血/再灌注损伤（IRI）可导致心力衰竭，这与焦亡有关，焦亡是一种促炎性程序性细胞死亡。已表明内源性小非编码RNA参与心肌IRI。在本研究中，我们旨在研究miR-424是否响应心肌IRI调节焦亡，并确定其潜在的调控机制。建立了心脏IRI小鼠模型，并通过超声心动图评估收缩功能。再灌注24小时后采集血清和心脏组织以评估焦亡状态。对于该研究，将H9C2细胞（一种大鼠心脏细胞系）置于缺氧6小时，然后再进行18小时复氧。通过实时PCR评估mRNA水平的基因表达，并通过蛋白质印迹、免疫荧光染色和酶联免疫吸附测定（ELISA）检测蛋白质水平的表达。应用生物信息学分析预测miR-424的靶标，然后通过荧光素酶报告基因测定进行确认。我们发现，心肌IRI时，包括半胱天冬酶-1、半胱天冬酶-11、白细胞介素-1β和白细胞介素-18在内的焦亡相关蛋白的表达显著增加。同样，缺氧/复氧损伤（HRI）也诱导H9C2细胞发生焦亡。此外，我们的研究表明，miR-424在I/R心脏组织和H/R刺激的H9C2细胞中的表达大幅增加。此外，我们发现miR-424的外源性表达直接靶向富含半胱氨酸的分泌蛋白LCCL结构域包含2（CRISPLD2），并上调半胱天冬酶-1以及促炎细胞因子白细胞介素-1β和白细胞介素-18的表达。综上所述，我们的研究结果提供了一种在IRI条件下心脏焦亡中miR-424/CRISPLD2的新信号通路。
