长链非编码 RNA PVT1/miR-590-5p/FSTL1 轴调节哮喘气道平滑肌细胞的增殖和迁移。
The lncRNA PVT1/miR-590-5p/FSTL1 axis modulates the proliferation and migration of airway smooth muscle cells in asthma.
机构信息
Department of Pediatrics, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, P.R. China.
出版信息
Autoimmunity. 2021 May;54(3):138-147. doi: 10.1080/08916934.2021.1897977. Epub 2021 Apr 7.
OBJECTIVE
Asthma is a prevalent chronic inflammatory airway disease that is characterised by airway remodelling and airway hyperresponsiveness. Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) contribute to airway remodelling in asthma. However, the molecular mechanism underlying an increased ASMC mass in asthma remains elusive. Herein, we aimed at investigating the regulation of lncRNA PVT1 on ASMCs and focussing on the mechanism in the proliferation and migration.
METHODS
Expression levels of lncRNA PVT1 and miR-590-5p in the serum collected from 24 children with asthma and 10 control children were determined by qRT-PCR. ASMCs proliferation and migration prior to and post platelet-derived growth factor subunit B (PDGF-BB) stimulation were examined by CCK-8 test and transwell assay. Dual-luciferase reporter assay was performed to determine miR-590-5p interaction with lncRNA PVT1 and follistatin-like 1 (FSTL1). Expression of lncRNA PVT1, miR-590-5p, FSTL1, C-Myc, cyclin D1, and cyclin-dependent kinase 1 (CDK1) was tested by quantitative real-time PCR (qRT-PCR) and immunoblotting analysis.
RESULTS
The expression level of lncRNA PVT1 was higher but the expression level of miR-590-5p was lower in the serum of children with asthma than in control children. The expression level of lncRNA PVT1 was negatively correlated with the expression level of miR-590-5p in asthma. LncRNA PVT1 was upregulated upon PDGF-BB stimulation. LncRNA PVT1 knockdown by its specific shRNA repressed PDGF-BB-induced promotion of proliferation and migration in ASMCs and triggered an elevated miR-590-5p along with declined C-Myc, cyclin D1, and CDK1. The effects of lncRNA PVT1 knockdown on PDGF-BB-induced ASMCs were lost upon miR-590-5p inhibition. MiR-590-5p targeted FSTL1 gene and declined its expression, thus suppressing ASMC proliferation and migration following PDGF-BB stimulation and downregulating C-Myc, cyclin D1, and CDK1 expressions. The effects of miR-590-5p on PDGF-BB-induced ASMCs were lost upon FSTL1 overexpression.
CONCLUSION
These results support the notion that the lncRNA PVT1/miR-590-5p/FSTL1 axis modulates ASMCs proliferation and migration following PDGF-BB stimulation, providing a potential therapeutic target to attenuate airway remodelling in asthma.
目的
哮喘是一种常见的慢性炎症性气道疾病，其特征为气道重塑和气道高反应性。气道平滑肌细胞（ASMCs）的异常增殖和迁移导致哮喘中的气道重塑。然而，哮喘中 ASMC 质量增加的分子机制仍不清楚。在此，我们旨在研究长链非编码 RNA PVT1 对 ASMC 的调节作用，并专注于增殖和迁移的机制。
方法
通过 qRT-PCR 测定来自 24 名哮喘儿童和 10 名对照儿童的血清中长链非编码 RNA PVT1 和 miR-590-5p 的表达水平。通过 CCK-8 试验和 Transwell 测定血小板衍生生长因子亚基 B（PDGF-BB）刺激前后 ASMC 的增殖和迁移。通过双荧光素酶报告基因测定确定 miR-590-5p 与长链非编码 RNA PVT1 和卵泡抑素样 1（FSTL1）的相互作用。通过定量实时 PCR（qRT-PCR）和免疫印迹分析检测长链非编码 RNA PVT1、miR-590-5p、FSTL1、C-Myc、细胞周期蛋白 D1 和细胞周期蛋白依赖性激酶 1（CDK1）的表达。
结果
与对照组儿童相比，哮喘儿童血清中的长链非编码 RNA PVT1 表达水平较高，而 miR-590-5p 表达水平较低。哮喘中长链非编码 RNA PVT1 的表达水平与 miR-590-5p 的表达水平呈负相关。PDGF-BB 刺激后长链非编码 RNA PVT1 上调。其特异性 shRNA 下调长链非编码 RNA PVT1 抑制了 ASMC 增殖和迁移，并触发 miR-590-5p 升高以及 C-Myc、细胞周期蛋白 D1 和 CDK1 降低。miR-590-5p 抑制后，长链非编码 RNA PVT1 敲低对 PDGF-BB 诱导的 ASMC 作用丧失。miR-590-5p 靶向 FSTL1 基因并降低其表达，从而抑制 PDGF-BB 刺激后的 ASMC 增殖和迁移，并下调 C-Myc、细胞周期蛋白 D1 和 CDK1 的表达。FSTL1 过表达后，miR-590-5p 对 PDGF-BB 诱导的 ASMC 的作用丧失。
结论
这些结果支持这样的观点，即长链非编码 RNA PVT1/miR-590-5p/FSTL1 轴调节 PDGF-BB 刺激后的 ASMC 增殖和迁移，为减轻哮喘中的气道重塑提供了一个潜在的治疗靶点。
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