环状 RNA BBS9 通过 miRNA-103a-3p/BCL2L13 在慢性阻塞性肺疾病中发挥重要作用。
Significant role of circRNA BBS9 in chronic obstructive pulmonary disease via miRNA-103a-3p/BCL2L13.
机构信息
Department of Pulmonary and Critical Care Medicine, Wuhan Fourth Hospital, Wuhan, 430030, China.
出版信息
BMC Pulm Med. 2023 Jul 13;23(1):257. doi: 10.1186/s12890-023-02540-2.
BACKGROUND
Various studies have shown that circular RNA (circRNA) plays a pivotal role in chronic obstructive pulmonary disease (COPD). We aimed to determine the role of circRNA BBS9 in COPD progression.
METHODS
Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to determine the levels and the linkages of circRNA BBS9, miRNA-103a-3p, and BCL2L13 in cigarette smoke extract (CSE)-treated human pulmonary microvascular endothelial cells (HPMECs). The target binding sites of circRNA BBS9 and miRNA-103a-3p were predicted using the starBase database, and the TargetScan algorithm was used to forecast the potential binding sites of BCL2L13 and miRNA-103a-3p, which were verified using a dual-luciferase reporter assay. An flow cytometry (FCM) assay was performed to determine the rate of apoptosis of HPMECs. Caspase3 activity was determined using a Caspase3 assay kit. The apoptosis-related protein bands were determined by western blotting.
RESULTS
The level of circRNA BBS9 increased in 1% CSE-induced cells, and silencing of circRNA BBS9 decreased the ratio of apoptotic cells among the 1% CSE-induced HPMECs. The results of dual-luciferase reporter assays showed that miRNA-103a-3p associates with circRNA BBS9. miRNA-103a-3p was downregulated in COPD, and upregulation of miRNA-103a-3p inhibited apoptosis in CSE-stimulated cells. Moreover, BCL2L13 was found to act downstream of miRNA-103a-3p. Silencing of miRNA-103a-3p reversed the inhibitory effect of circRNA BBS9-siRNA. The effects of the miRNA-103a-3p mimic were reversed by the BCL2L13-plasmid.
CONCLUSION
circRNA BBS9 is involved in COPD development as it inhibits the functioning of miRNA-103a-3p. Our results suggest that circRNA BBS9 may act as a novel target for treating COPD.
背景
多项研究表明，环状 RNA（circRNA）在慢性阻塞性肺疾病（COPD）中发挥着关键作用。我们旨在确定 circRNA BBS9 在 COPD 进展中的作用。
方法
通过实时定量逆转录 PCR（qRT-PCR）来确定香烟烟雾提取物（CSE）处理的人肺微血管内皮细胞（HPMEC）中 circRNA BBS9、miRNA-103a-3p 和 BCL2L13 的水平和关联性。circRNA BBS9 和 miRNA-103a-3p 的靶标结合位点使用 starBase 数据库进行预测，使用 TargetScan 算法预测 BCL2L13 和 miRNA-103a-3p 的潜在结合位点，并通过双荧光素酶报告基因检测进行验证。通过流式细胞术（FCM）检测 HPMEC 凋亡率。通过 Caspase3 测定试剂盒测定 Caspase3 活性。通过 Western blot 检测凋亡相关蛋白条带。
结果
在 1% CSE 诱导的细胞中 circRNA BBS9 的水平增加，沉默 circRNA BBS9 降低了 1% CSE 诱导的 HPMEC 中凋亡细胞的比例。双荧光素酶报告基因检测结果表明，miRNA-103a-3p 与 circRNA BBS9 结合。COPD 中 miRNA-103a-3p 下调，上调 miRNA-103a-3p 可抑制 CSE 刺激的细胞凋亡。此外，发现 BCL2L13 是 miRNA-103a-3p 的下游靶点。沉默 miRNA-103a-3p 逆转了 circRNA BBS9-siRNA 的抑制作用。miRNA-103a-3p 模拟物的作用被 BCL2L13 质粒逆转。
结论
circRNA BBS9 通过抑制 miRNA-103a-3p 的功能参与 COPD 的发生发展。我们的研究结果表明，circRNA BBS9 可能成为治疗 COPD 的新靶点。
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