circNFXL1 Modulates the Kv2.1 Channel Function in Hypoxic Human Pulmonary Artery Smooth Muscle Cells via Sponging miR-29b-2-5p as a Competitive Endogenous RNA

Pulmonary arterial hypertension is characterized by abnormal pulmonary vasoconstriction and vascular remodeling caused by the dysregulation of K(+) channels in PA smooth muscle cells (PASMCs). However, how the K(+) channels are dysregulated is still unclear. Circular RNAs (circRNAs) are noncoding RNAs with a closed-loop structure capable of sponging microRNAs (miRs), thus regulating gene expression at the post-transcriptional level. Our previous studies have demonstrated the importance of one novel circRNA (hsa_circNFXL1_009, circNFXL1) in pulmonary arterial hypertension patients, playing as a critical regulator for K(+) channel activation in hypoxic human PASMCs (hPASMCs). Here, we explore the mechanisms underlying circNFXL1-regulated K(+) channel expression and functions in hypoxic hPASMCs. In cultured hPASMCs, the reduction of Kv current induced by hypoxia was significantly recovered by delivering exogenous circNFXL1. Moreover, luciferase, quantitative reverse transcription-quantitative polymerase chain reaction, western blot, and mutagenesis studies confirmed that circNFXL1 reversed hypoxia-induced inhibitory effects on the Kv2.1 channel via sponging hsa-miR-29b-2-5p (miR-29b-2). Furthermore, we found that circNFXL1 reversed the miR-29b-induced Kv2.1 channel dysfunction at the whole-cell and single-channel level in HEK cells using a patch-clamp. Finally, calcium imaging revealed that hypoxia also triggered a substantial rise in the cytosolic calcium concentration ([Ca2(+)]cyt) in hPASMCs, and this hypoxia-induced elevation of [Ca2(+)]cyt was reduced by circNFXL1 through miR-29b-2. These data suggested that circNFXL1-mediated regulation of the Kv2.1 channel activation and the related intracellular calcium concentration may contribute to the effects of hypoxic pulmonary vasoconstriction.