Dataset Information

CircHECTD1 mediates pulmonary fibroblast activation via HECTD1.

ABSTRACT:

Background

Circular RNA (circRNA), a new class of noncoding RNA, has been shown to be important in silicosis due to its unique role as a transcription regulator or as a sponge of small RNA regulators. Here, the mechanisms underlying circHECTD1/HECTD1 in fibroblast activation and subsequent fibrosis induced by SiO₂ were investigated.

Methods

Primary human pulmonary fibroblasts (HPF-a) were utilized, combined with quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) assays. LC3B-LV-RFP lentivirus was used to evaluate the role of autophagy. The CRISPR/Cas9 system was applied to specifically knock down HECTD1, combined with MTT, BrdU, and migration assays, to explore the functional changes induced by SiO₂.

Results

After exposure to SiO₂, the circHECTD1 level was decreased, which was associated with an increase in HECTD1 in HPF-a cells. SiO₂-induced autophagy was reversed by either circHECTD1 overexpression or HECTD1 knockdown in HPF-a cells, with restored SiO₂-induced fibroblast activation, proliferation, and migration via downstream autophagy. The lungs of mice exposed to SiO₂ confirmed the upregulation of HECTD1 in pulmonary fibroblasts.

Conclusions

Our data suggested a link between circHECTD1/HECTD1 and fibroblast activation with subsequent fibrosis induced by SiO₂, providing novel insight into the potential of circHECTD1/HECTD1 to be a therapeutic target for silicosis.

SUBMITTER: Chu H

PROVIDER: S-EPMC6887829 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

CircHECTD1 mediates pulmonary fibroblast activation <i>via</i> HECTD1.

Chu Han H Wang Wei W Luo Wei W Zhang Wei W Cheng Yusi Y Huang Jie J Wang Jing J Dai Xiaoniu X Fang Shencun S Chao Jie J

Therapeutic advances in chronic disease 20191127

<h4>Background</h4>Circular RNA (circRNA), a new class of noncoding RNA, has been shown to be important in silicosis due to its unique role as a transcription regulator or as a sponge of small RNA regulators. Here, the mechanisms underlying circHECTD1/HECTD1 in fibroblast activation and subsequent fibrosis induced by SiO<sub>2</sub> were investigated.<h4>Methods</h4>Primary human pulmonary fibroblasts (HPF-a) were utilized, combined with quantitative real-time PCR (qRT-PCR) and fluorescence in s ...[more]

PMID: 31832126

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Project description:Excessive proliferation and migration of fibroblasts contribute to pulmonary fibrosis in silicosis, and both epithelial cells and endothelial cells participate in the accumulation of fibroblasts via the epithelial-mesenchymal transition (EMT) and the endothelial-mesenchymal transition (EndMT), respectively. A mouse endothelial cell line (MML1) was exposed to silicon dioxide (SiO2, 50 μg/cm2), and immunofluorescence and western blot analyses were performed to evaluate levels of specific endothelial and mesenchymal markers and to elucidate the mechanisms by which SiO2 induces the EndMT. Functional changes were evaluated by analyzing cell migration and proliferation. The mRNA and circular RNA (circRNA) levels were measured using qPCR and fluorescent in situ hybridization (FISH). Lung tissue samples from both Tie2-GFP mice exposed to SiO2 and silicosis patients were applied to confirm the observations from in vitro experiments. Based on the results from the current study, SiO2 increased the expression of mesenchymal markers (type I collagen (COL1A1), type III collagen (COL3A1) and alpha smooth muscle actin (α-SMA/Acta2)) and decreased the expression of endothelial markers (vascular endothelial cadherin (VE-Cad/Cdh 5) and platelet endothelial cell adhesion molecule-1 (PECAM1)), indicating the occurrence of the EndMT in response to SiO2 exposure both in vivo and in vitro. SiO2 concomitantly increased circHECTD1 expression, which, in turn, inhibited HECTD1 protein expression. SiO2-induced increases in cell proliferation, migration, and changes in marker levels were restored by either a small interfering RNA (siRNA) targeting circHECTD1 or overexpression of HECTD1 via the CRISPR/Cas9 system, confirming the involvement of the circHECTD1/HECTD1 pathway in the EndMT. Moreover, tissue samples from SiO2-exposed mice and silicosis patients confirmed the EndMT and change in HECTD1 expression. Our findings reveal a potentially new function for the circHECTD1/HECTD1 pathway and suggest a possible mechanism of fibrosis in patients with pulmonary silicosis.