Background

Circular RNAs (circRNAs) function as important modulators in the progression of pulmonary arterial hypertension (PAH). Here, we aimed to discover the role and working mechanism of circATP2B4 in hypoxia-induced proliferation and migration of PASMCs.

Methods

The proliferation, migration and apoptosis of pulmonary arterial smooth muscle cells (PASMCs) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound healing assay and flow cytometry. The expression of circATP2B4, ATPase plasma membrane Ca²⁺ transporting 4 (ATP2B4), microRNA-223 (miR-223) and ATR serine/threonine kinase (ATR) was quantified by quantitative real time polymerase chain reaction (qRT-PCR). Circular RNA Interactome and microT-CDS were used to search the targets of circATP2B4 and miR-223, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to validate the above predictions. Western blot assay was performed to detect the protein expression of ATR.

Results

Hypoxia treatment promoted the proliferation and migration and impeded the apoptosis of PASMCs. A significant increase of circATP2B4 expression was observed in the serum of PAH patients and hypoxia-induced PASMCs compared with healthy volunteers and PASMCs under normoxia condition. MiR-223 is a target of circATP2B4, and the effects of circATP2B4 silencing on PASMCs were overturned by the transfection of anti-miR-223. ATR is a functional target of miR-223, and miR-223 inhibited the proliferation and migration while accelerated the apoptosis of PASMCs through targeting and down-regulating ATR. CircATP2B4 could up-regulate the level of ATR through sponging miR-223 in PSAMCs.

Conclusion

CircATP2B4 potentiated hypoxia-induced proliferation and migration of PASMCs through the miR-223/ATR axis. Restoration of the level of miR-223 might be an effective therapeutic method for the treatment of PAH.

中文翻译：

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CircATP2B4通过miR-223 / ATR轴促进低氧诱导的肺动脉平滑肌细胞增殖和迁移。

背景

环状RNA（circRNA）在肺动脉高压（PAH）的进程中起着重要的调节剂的作用。在这里，我们旨在发现circATP2B4在缺氧诱导的PASMCs增殖和迁移中的作用和工作机制。

方法

肺动脉平滑肌细胞（PASMCs）的增殖，迁移和凋亡通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑鎓溴化物（MTT），伤口愈合测定和流式细胞仪检测。通过定量实时聚合酶链反应（qRT-PCR）定量检测circATP2B4，ATP酶质膜Ca ²⁺转运4（ATP2B4），microRNA-223（miR-223）和ATR丝氨酸/苏氨酸激酶（ATR）的表达。使用环形RNA相互作用组和microT-CDS分别搜索circATP2B4和miR-223的靶标。双萤光素酶报告基因测定和RNA免疫沉淀（RIP）测定用于验证上述预测。进行蛋白质印迹分析以检测ATR的蛋白表达。

结果

低氧处理促进了PASMCs的增殖和迁移，并阻止了其凋亡。与正常志愿者和常氧条件下的PASMCs相比，PAH患者和低氧诱导的PASMCs的血清中circATP2B4表达显着增加。MiR-223是circATP2B4的靶标，而抗miR-223的转染可消除circATP2B4沉默对PASMC的作用。ATR是miR-223的功能靶标，并且miR-223通过靶向和下调ATR抑制PASMCs的增殖和迁移，同时促进其凋亡。CircATP2B4可以通过使PSAMC中的miR-223海绵化而上调ATR的水平。

结论

CircATP2B4增强了缺氧诱导的PASMC通过miR-223 / ATR轴的增殖和迁移。恢复miR-223的水平可能是治疗PAH的有效治疗方法。