Circ_0035796 耗竭以 miR-150-5p/L1CAM 依赖性方式抑制转化生长因子-β1 诱导的肺纤维化。
Circ_0035796 depletion inhibits transforming growth factor-β1-induced pulmonary fibrosis in a miR-150-5p/L1CAM-dependent manner.
机构信息
Department of Clinical Laboratory, Beijing Friendship Hospital, Capital Medical University, Beijing City, P.R. China.
出版信息
Autoimmunity. 2023 Dec;56(1):2250099. doi: 10.1080/08916934.2023.2250099. Epub 2023 Oct 11.
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 .
背景
肺纤维化的发病机制尚不完全清楚。先前的研究表明，环状 RNA（circRNA）在肺纤维化的发展中起着重要作用。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）处理人胚肺成纤维细胞 1（HFL1）细胞，模拟肺纤维化细胞模型。采用实时定量聚合酶链反应（qRT-PCR）检测 circ_0035796、微小 RNA-150-5p（miR-150-5p）和 L1 细胞黏附分子（L1CAM）的表达。采用 Western blot 检测 L1CAM、胶原 I 和纤连蛋白的蛋白表达。用 CCK-8 法检测细胞活力。用 5-乙炔基-2'-脱氧尿苷（EdU）检测、Transwell 侵袭实验和划痕愈合实验分别检测细胞增殖、侵袭和迁移。采用酶联免疫吸附试验（ELISA）检测白细胞介素 6（IL-6）和肿瘤坏死因子-α（TNF-α）的分泌。采用商业试剂盒检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平评估氧化应激。通过双荧光素酶报告基因实验、RNA 下拉实验和 RNA 免疫沉淀（RIP）实验鉴定 miR-150-5p 与 circ_0035796 和 L1CAM 的关联。
结果
TGF-β1 处理的 HFL1 细胞中 circ_0035796 和 L1CAM 表达明显上调，而 miR-150-5p 表达下调。TGF-β1 处理诱导细胞增殖、迁移、侵袭、IL-6 和 TNF-α分泌以及氧化应激，而 circ_0035796 耗竭则缓解了这些作用。此外，circ_0035796 作为 miR-150-5p 的海绵，miR-150-5p 与 L1CAM 结合。此外，miR-150-5p 耗竭可减轻 TGF-β1 暴露的 HFL1 细胞中 circ_0035796 敲低介导的作用。miR-150-5p 对 TGF-β1 诱导的成纤维细胞激活的调节涉及 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 表达。
结论
circ_0035796 敲低通过 miR-150-5p/L1CAM 轴减轻 TGF-β1 诱导的肺纤维化。
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