miR-424 promotes cardiac ischemia / reperfusion injury by direct targeting of CRISPLD2 and regulating cardiomyocyte pyroptosis

As a complex pathophysiological event, myocardial ischemia/reperfusion injury (IRI) can cause heart fail- ure, 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 mech- anism. An in vivo mouse model of cardiac IRI was established, and contractile function was evaluated by echogra- phy. The serum and heart tissue were harvested 24 h after reperfusion to assess the status of pyroptosis. For the in vitro 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 expres- sions 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.