Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis Via the Circ-Calm4/miR-124-3p/PDCD6 Axis

Objective: Circular RNAs are emerging as important regulators of pulmonary hypertension where pyroptosis plays a critical role. However, whether and how the circular RNAs regulate pyroptosis remained unexplored. Here, we show evidence for the involvement of a specific circular RNA known as circ-Calm4 in pulmonary hypertension and the underlying signaling pathway in pyroptosis. Approach and Results: Circ-Calm4 was upregulated in both mouse model of pulmonary hypertension in vivo and cultured smooth muscle cells in vitro. We performed immunoblotting, quantitative real-time PCR, LDH (lactate dehydrogenase) release assay, Annexin V-FITC/propidium iodide double staining, Hoechst 33342/propidium iodide fluorescence staining, and immunostaining to clarify the roles of circ-Calm4 in pulmonary arterial smooth muscle cell pyroptosis. Silencing the circ-Calm4 with its small-interfering RNA mitigated the upregulation of pyroptosis related phenotypes induced by hypoxia. Luciferase reporter assays confirmed that miR-124-3p suppressed the luciferase activity of the circ-Calm4 and RNA fluorescence in situ hybridization showed the colocalization of circ-Calm4 and miR-124-3p. The circ-Calm4 was found to act as a competitive endogenous RNA to regulate miR-124-3p. The pyroptosis-related alterations were all diminished with miR-124-3p in hypoxic pulmonary arterial smooth muscle cells. Inhibition of the gene targeted by miR-124-3p encoding the Pdcd6 (programmed cell death protein 6) abrogated pyroptosis-related phenotypes under hypoxia stimulation. Conclusions: Our findings show a new signaling pathway, the circ-Calm4/miR-124-3p/Pdcd6 axis was demonstrated in regulation of hypoxia-induced pyroptosis, which may potentially be useful for the design of therapeutic strategies for protecting the cellular functionality against pyroptosis as well as pulmonary hypertension.