Circ_0035796 depletion inhibits transforming growth factor-β1-induced pulmonary fibrosis in a miR-150-5p/L1CAM-dependent manner.

IF 3.3 4区医学 Q3 IMMUNOLOGY Autoimmunity Pub Date : 2023-12-01 Epub Date: 2023-10-11 DOI:10.1080/08916934.2023.2250099

Juan Li, Xiaohong Chen, Baohong Zhang, Chenlu Wang

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Abstract

Background: The pathogenesis of pulmonary fibrosis is not fully understood. Previous work has demonstrated the important role of circular RNA (circRNA) in pulmonary fibrosis development. This study aims to analyse the role of circ_0035796 in pulmonary fibrosis and the underlying mechanism.

Methods: Human foetal lung fibroblast 1 (HFL1) cells were treated with transforming growth factor-β1 (TGF-β1) to mimic a pulmonary fibrosis cell model. The expression of circ_0035796, microRNA-150-5p (miR-150-5p) and L1 cell adhesion molecule (L1CAM) was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of L1CAM, collagen I and fibronectin was detected by Western blot. Cell viability was analysed by CCK-8 assay. Cell proliferation, invasion and migration were investigated by 5-Ethynyl-2'-deoxyuridine (EdU) assay, transwell invasion assay and wound-healing assay, respectively. The secretion of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) was analysed by Enzyme-linked immunosorbent assay (ELISA). Oxidative stress was assessed by detecting Superoxide Dismutase (SOD) activity and Malondialdehyde (MDA) level using commercial kits. The association of miR-150-5p with circ_0035796 and L1CAM was identified by dual-luciferase reporter assay, RNA pull-down assay and RNA immunoprecipitation (RIP) assay.

Results: Circ_0035796 and L1CAM expression were dramatically upregulated, while miR-150-5p expression was downregulated in TGF-β1-treated HFL1 cells. TGF-β1 treatment induced cell proliferation, migration, invasion, IL-6 and TNF-α secretion, and oxidative stress, whereas circ_0035796 depletion relieved these effects. In addition, circ_0035796 acted as a sponge of miR-150-5p and miR-150-5p combined with L1CAM. Moreover, miR-150-5p depletion attenuated circ_0035796 knockdown-mediated effects in TGF-β1-exposed HFL1 cells. The regulation of miR-150-5p on TGF-β1-induced fibroblast activation involved the downregulation of L1CAM. Further, circ_0035796 modulated L1CAM expression by interacting with miR-150-5p in TGF-β1-exposed HFL1 cells.

Conclusion: Circ_0035796 knockdown ameliorates TGF-β1-induced pulmonary fibrosis through the miR-150-5p/L1CAM axis in vitro.

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Circ_0035796耗竭以miR-150-5p/L1CAM依赖的方式抑制转化生长因子-β1诱导的肺纤维化。

背景：肺纤维化的发病机制尚不完全清楚。先前的工作已经证明了环状RNA（circRNA）在肺纤维化发展中的重要作用。本研究旨在分析circ_0035796在肺纤维化中的作用及其潜在机制。方法：用转化生长因子-β1（TGF-β1）处理人胎肺成纤维细胞1（HFL1），模拟肺纤维化细胞模型。通过定量实时聚合酶链式反应（qRT-PCR）测定circ_0035796、microRNA-150-5p（miR-150-5p）和L1细胞粘附分子（L1CAM）的表达。蛋白质印迹法检测L1CAM、I型胶原和纤连蛋白的表达。通过CCK-8测定法分析细胞活力。分别用5-乙炔基-2'-脱氧尿苷（EdU）法、transwell侵袭法和伤口愈合法研究细胞增殖、侵袭和迁移。采用酶联免疫吸附法（ELISA）检测白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的分泌。使用商业试剂盒通过检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平来评估氧化应激。miR-150-5p与circ_0035796和L1CAM的关联通过双荧光素酶报告基因测定、RNA下拉测定和RNA免疫沉淀（RIP）测定来鉴定。结果：在TGF-β1处理的HFL1细胞中，Circ_0035796和L1CAM的表达显著上调，而miR-150-5p的表达下调。TGF-β1治疗诱导了细胞增殖、迁移、侵袭、IL-6和TNF-α分泌以及氧化应激，而circ_0035796的耗竭减轻了这些影响。此外，circ_0035796充当miR-150-5p和miR-150-5p与L1CAM结合的海绵。此外，miR-150-5p缺失减弱了暴露于TGF-β1的HFL1细胞中circ_0035796敲低介导的作用。miR-150-5p对TGF-β1诱导的成纤维细胞活化的调节涉及L1CAM的下调。此外，circ_0035796通过与TGF-β1暴露的HFL1细胞中的miR-150-5p相互作用来调节L1CAM的表达。结论：Circ_0035796敲低通过miR-150-5p/L1CAM轴在体外改善TGF-β1诱导的肺纤维化。

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来源期刊

Autoimmunity 医学-免疫学

CiteScore

5.70

自引率

8.60%

发文量

审稿时长

6-12 weeks

期刊介绍： Autoimmunity is an international, peer reviewed journal that publishes articles on cell and molecular immunology, immunogenetics, molecular biology and autoimmunity. Current understanding of immunity and autoimmunity is being furthered by the progress in new molecular sciences that has recently been little short of spectacular. In addition to the basic elements and mechanisms of the immune system, Autoimmunity is interested in the cellular and molecular processes associated with systemic lupus erythematosus, rheumatoid arthritis, Sjogren syndrome, type I diabetes, multiple sclerosis and other systemic and organ-specific autoimmune disorders. The journal reflects the immunology areas where scientific progress is most rapid. It is a valuable tool to basic and translational researchers in cell biology, genetics and molecular biology of immunity and autoimmunity.