circHECTD1 promotes the silica-induced pulmonary endothelial–mesenchymal transition via HECTD1

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.