School of Medicine, Nankai University, Tianjin, China.
Tianjin Central Hospital of Gynecology Obstetrics, Tianjin, China.
J Cardiovasc Pharmacol. 2023 Apr 1;81(4):292-299. doi: 10.1097/FJC.0000000000001396.
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.
肺动脉高压的特征是肺血管平滑肌细胞 (PASMC) 中 K + 通道的失调引起的异常肺血管收缩和血管重塑。然而，K + 通道如何失调仍不清楚。环状 RNA（circRNA）是具有闭合环结构的非编码 RNA，能够海绵状 microRNA（miR），从而在转录后水平调节基因表达。我们之前的研究表明，一种新型环状 RNA（hsa_circNFXL1_009，circNFXL1）在肺动脉高压患者中的重要性，作为缺氧人 PASMCs（hPASMCs）中 K + 通道激活的关键调节剂。在这里，我们探讨了 circNFXL1 调节缺氧诱导的 hPASMCs 中 K + 通道表达和功能的机制。在培养的 hPASMCs 中，外源性 circNFXL1 的传递显著恢复了由缺氧引起的 Kv 电流减少。此外，荧光素酶、定量逆转录-定量聚合酶链反应、western blot 和突变研究证实，circNFXL1 通过海绵状 hsa-miR-29b-2-5p（miR-29b-2）逆转缺氧对 Kv2.1 通道的抑制作用。此外，我们发现 circNFXL1 在 HEK 细胞中通过整细胞和单通道记录逆转了 miR-29b 诱导的 Kv2.1 通道功能障碍。最后，钙成像显示，缺氧也会引起 hPASMCs 胞质钙离子浓度 ([Ca2 + ] cyt) 的大幅升高，circNFXL1 通过 miR-29b-2 降低了这种缺氧诱导的 [Ca2 + ] cyt 升高。这些数据表明，circNFXL1 介导的 Kv2.1 通道激活和相关细胞内钙浓度的调节可能有助于缺氧性肺血管收缩的作用。
