[Mechanism of SMURF2 regulating silicosis-associated epithelial-mesenchymal transition via the TGF-β/Smad signaling...

Objective: To explore the mechanism by which targeted deletion of Smad ubiquitin regulatory factor 2 (SMURF2/Smurf2) in alveolar type Ⅱ epithelial cells (AT2) inhibits epithelial-mesenchymal transition (EMT) in silicosis. Methods: In March 2024, in vitro experiments were conducted using a mouse AT2 cell line (MLE-12), induced by silicon dioxide (SiO(2)), and small interfering RNA (siRNA) was employed for Smurf2 gene silencing. The cells were divided into control group, SiO(2) group (250 μg/ml), empty vector plasmid (siRNA-NC) +SiO(2) group (first transfected with siRNA-NC for 24 hours and then induced with SiO(2) for 24 hours), and si-Smurf2+SiO(2) group (first transfected with si-Smurf2 for 24 hours and then induced with SiO(2) for 24 hours). In vivo experiments, 20 SPF-grade C57BL/6J mice with targeted AT2 cell Smurf2 gene knockout (Smurf2(-/-)) at 8 weeks of age were randomly divided into Smurf2(-/-) control group and Smurf2(-/-) silicosis group, with 10 mice in each group; another 20 control mice were randomly divided into Smurf2(+/+) control group and Smurf2(+/+) silicosis group, with 10 mice in each group. Mice in Smurf2(-/-) silicosis group and Smurf2(+/+) silicosis group received a single intratracheal instillation of SiO(2) suspension (5 mg per mouse, 50 μl), while the remaining groups were instilled with an equal volume of normal saline. All mice were maintained until 4 weeks post-instillation. Hematoxylin eosin (HE) staining was used to observe the pathological morphology of the lung tissue, Masson staining was used to observe the distribution and deposition of collagen, and micro-CT was used to reconstruct the three-dimensional structure of the lung. Immunofluorescence staining was used to detect the co-localization of E-cadherin (E-Ca) /alpha smooth muscle actin (α-SMA) and pro-surfactant protein C (pro-SPC) /SMURF2. Western blotting was used to quantitatively analyze the expression of SMURF2, transforming growth factor beta (TGF-β) signaling-related proteins, E-Ca, N-cadherin (N-Ca), and α-SMA. Co-immunoprecipitation was used to detect the binding effect between SMURF2 and Smad7, and in vitro ubiquitination assay was used to detect the ubiquitination of Smad7. One-way ANOVA was used for comparisons among multiple groups, and LSD test was used for pairwise comparisons when variances were homogeneous, and Tamhane's T2 test was used when variances were not homogeneous. Results: Compared with the control group, the expressions of E-Ca and Smad7 were downregulated in MLE-12 cells of the SiO(2) group, while the expressions of N-Ca, α-SMA, TGF-β1, TGF-β receptor 1 (TGF-βR1), TGF-β receptor 2 (TGF-βR2), and phosphorylated Smad2/3 (p-Smad2/3) were upregulated (P<0.05) ; SMURF2 bound to Smad7, and Smad7 bound to ubiquitin (Ub). Compared with the siRNA-NC+SiO(2) group, the expressions of E-Ca and Smad7 were upregulated in the si-Smurf2+SiO(2) group, while the expressions of N-Ca, α-SMA, TGF-β1, TGF-βR1, TGF-βR2, and p-Smad2/3 were downregulated (P<0.05), the binding of Smad7 to Ub was inhibited. Compared with the Smurf2(+/+) control group, the formation of silica nodules and collagen deposition occurred simultaneously in Smurf2(+/+) silicosis mice, and the volume of high-density shadows in the lung tissue increased, with downregulated expressions of E-Ca, Smad7, and upregulated expressions of N-Ca, α-SMA, TGF-β1, TGF-βR1, TGF-βR2, and p-Smad2/3 (P<0.05). Compared with the Smurf2(+/+) silicosis group, the area of silica nodules and collagen deposition decreased in the Smurf2(-/-) silicosis group, with upregulated expressions of E-Ca, Smad7, and downregulated expressions of N-Ca, α-SMA, TGF-β1, TGF-βR1, TGF-βR2, and p-Smad2/3 (P<0.05) . Conclusion: Targeted knockout of the Smurf2 gene in AT2 cells may inhibit EMT by regulating the TGF-β/Smad signaling pathway, thereby delaying the progression of silicosis-induced pulmonary fibrosis.