Abstract

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.

中文翻译：

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lncRNA PVT1/miR-590-5p/FSTL1轴调节哮喘气道平滑肌细胞的增殖和迁移

摘要

客观的

哮喘是一种流行的慢性炎症性气道疾病，其特征是气道重塑和气道高反应性。气道平滑肌细胞 (ASMC) 的异常增殖和迁移有助于哮喘患者的气道重塑。然而，哮喘中 ASMC 质量增加的分子机制仍然难以捉摸。在此，我们旨在研究 lncRNA PVT1 对 ASMC 的调控，并重点研究其增殖和迁移的机制。

方法

采用qRT-PCR检测24例哮喘患儿和10例对照患儿血清中lncRNA PVT1和miR-590-5p的表达水平。通过 CCK-8 测试和 transwell 测定检查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。进行双荧光素酶报告基因测定以确定 miR-590-5p 与 lncRNA PVT1 和卵泡抑素样 1 (FSTL1) 的相互作用。通过实时定量 PCR (qRT-PCR) 和免疫印迹分析检测 lncRNA 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

哮喘患儿血清中lncRNA PVT1的表达水平高于对照组，而miR-590-5p的表达水平低于对照组。lncRNA PVT1的表达水平与哮喘中miR-590-5p的表达水平呈负相关。LncRNA PVT1 在 PDGF-BB 刺激下上调。LncRNA PVT1 被其特定的 shRNA 抑制抑制了 PDGF-BB 诱导的 ASMC 增殖和迁移的促进，并引发了 miR-590-5p 升高以及 C-Myc、细胞周期蛋白 D1 和 CDK1 的下降。lncRNA PVT1 敲低对 PDGF-BB 诱导的 ASMC 的影响在 miR-590-5p 抑制后消失。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。

结论

这些结果支持了 lncRNA PVT1/miR-590-5p/FSTL1 轴在 PDGF-BB 刺激后调节 ASMC 增殖和迁移的观点，为减轻哮喘气道重塑提供了潜在的治疗靶点。