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

Abstract

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.

Circ-BPTF Associated Pulmonary Vascular Remodeling in COPD Via Promoting Smooth Muscle Cells Proliferation by MiR-486-5p/CEMIP Axis

“…Although some reports have proved the role of circRNA in cardiovascular disorders and pulmonary hypertension, few studies focused on circRNA in COPD-associated pulmonary vascular remodeling (Yang et al 2020;Jiang et al 2021). In this study, we used microarray analysis to identify changes in the expression pro les of circRNAs in lung tissues of COPD patients and in PASMCs exposed to hypoxia.…”

Circular RNAs in pulmonary hypertension: Emerging biological concepts and potential mechanism

“… 71 Their subsequent study indicated that circ_Calm4 also acted as a ceRNA, regulating the expression of miR‐124‐3p/ PDCD6 to modulate the pyroptosis of hypoxia‐cultured PASMCs (Figure 3). 69 Moreover, Yang et al reported that mmu_circ_0000790 competitively bound to miR‐374c, thus upregulating FOXC1 expression and activating notch pathway in the pulmonary vascular tissues of HPH mice and hypoxia‐cultured PASMCs (Figure 3). 77 Guo et al reported that circATP2B4/miR‐223/ ATR axis regulated the proliferation, migration, and apoptosis of PASMCs induced by hypoxia 24 .…”

“…Using RNA seq, it has been demonstrated that circRNA levels generally increase or decrease in different types of PH. 69,70 Although the limited number of PH-related circRNAs studies to date have only focused on their functions as biomarkers and miRNA sponges, the data clearly indicate that circRNAs had a critical effect on the pathogenesis of PH, which are summarized in Figure 3. 46,71…”

Regulation of Pyroptosis by ncRNA: A Novel Research Direction

“…In the exploration of diabetic cardiomyopathy (DCM), Yang et al emphasized that hsa_circ_0076631, named caspase-1-associated circRNA (CACR), was significantly increased in the serum of diabetic patients, and the silencing of CACR could reduce caspase-1 expression by sponging miR-214-3p, thereby restraining inflammation and pyroptosis of cardiomyocytes ( Yang F. et al, 2019 ). Jiang et al proved that in hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs), circ-Calm4 increased the expression of programmed cell death protein 6 (PDCD6) through sponging miR-124-3p, and ultimately triggered pyroptosis ( Jiang et al, 2021 ). Although the current research on circRNA-related ceRNA in pyroptosis is insufficient, it is believed that the achievements in this field would be even greater in over time.…”