Effects of miR-204-3p inhibitor on epithelial-mesenchymal transition and silicosis fibrosis in silicon dioxide-induced alveolar epithelial cells
- VernacularTitle:miR-204-3p抑制剂对二氧化硅诱导的肺泡上皮细胞EMT过程与矽肺纤维化的影响
- Author:
Fang CHEN
1
;
Jing YU
1
;
Wenxuan HU
1
;
Yangyang PI
1
;
Xi ZHANG
1
;
Luning WANG
1
;
Ping ZHAO
1
;
Faxuan WANG
1
Author Information
- Publication Type:Experiment
- Keywords: silicon dioxide dust; alveolar epithelial cell; epithelial-mesenchymal transition; silicosis fibrosis; miR-204-3p
- From: Journal of Environmental and Occupational Medicine 2025;42(5):622-629
- CountryChina
- Language:Chinese
- Abstract: Background The pathogenesis of silicosis has not been fully elucidated, and microRNAs (miRNA) may be involved in the occurrence and development of silicosis. Objective To investigate the effect of miR-204-3p inhibitor on the epithelial-mesenchymal transition (EMT) process and silicosis fibrosis in silicon dioxide dust-induced alveolar epithelial cells. Methods A co-culture model of macrophages and epithelial cells was established using a Transwell chamber. NR8383 macrophages were seeded into the upper chamber of the Transwell, and RLE-6TN cells were seeded into the lower chamber. After 24 h of culture, the medium in the lower chamber was discarded, washed three times with phosphate-buffered saline (PBS), and replaced with serum-free medium. The cells were divided into four groups: control group, silicosis group, miRNA NC group, and miR-204-3p inhibitor group. The lower chamber was transfected with miRNA NC for the miRNA NC group or the miR-204-3p inhibitor for the miR-204-3p inhibitor group. The lower chambers of the remaining two groups were added by equal amounts of serum-free medium. After 24 h, except for the control group that received an equal volume of serum-free medium, the upper chambers of the remaining three groups were treated with 800 μg·mL−1 silicon dioxide dust. Morphological changes in each group were observed under a microscope. The mRNA and protein expression levels of EMT-related factors, including α-smooth muscle actin (α-SMA), Vimentin, N-Cadherin, and E-Cadherin, were detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot. The mRNA and protein expression levels of fibrosis-related factors, including Collagen I, Collagen III, and Fibronectin, were also assessed by RT-qPCR and Western blot. The fluorescence expression intensities of α-SMA, N-Cadherin, and E-Cadherin were evaluated by immunofluorescence. Results The morphological observation revealed that RLE-6TN cells in the control group exhibited a regular oval shape. After treatment with silicon dioxide, the cells predominantly displayed a long spindle shape. Following the intervention with the miR-204-3p inhibitor, the number of long spindle-shaped cells increased, and the intercellular gaps widened. The RT-qPCR results showed that, compared with the control group, the silicosis group exhibited significantly higher relative mRNA expression levels of EMT-related markers (α-SMA, Vimentin, and N-Cadherin) (P<0.05), while the relative mRNA expression level of E-Cadherin was significantly reduced (P<0.05); the relative mRNA expression levels of fibrosis-related markers (Collagen I, Collagen III, and Fibronectin) were also significantly elevated (P<0.05). Compared with the miRNA NC group, the miR-204-3p inhibitor group showed significantly increased relative mRNA expression levels of α-SMA, Vimentin, and N-Cadherin (P<0.05), decreased E-Cadherin mPNA expression (P<0.05), and elevated mPNA expression of Collagen I, Collagen III, and Fibronectin (P<0.05). The Western blot analysis indicated that, compared with the control group, the silicosis group had significantly higher protein expression levels of α-SMA, Vimentin, and N-Cadherin (P<0.05), lower E-Cadherin protein expression (P<0.05), and increased protein expression of Collagen I, Collagen III, and Fibronectin (P<0.05). Compared with the miRNA NC group, the miR-204-3p inhibitor group exhibited significantly elevated protein expression levels of α-SMA, Vimentin, and N-Cadherin (P<0.05), reduced E-Cadherin expression (P<0.05), and increased protein expression of Collagen I, Collagen III, and Fibronectin (P<0.05). The immunofluorescence analysis demonstrated that, compared with the control group, the silicosis group showed enhanced fluorescence intensities of α-SMA and N-Cadherin and reduced fluorescence intensity of E-Cadherin. Compared with the miRNA NC group, the miR-204-3p inhibitor group exhibited increased fluorescence intensities of α-SMA and N-Cadherin and decreased fluorescence intensity of E-Cadherin. Conclusion The miR-204-3p inhibitor may exacerbate the EMT process and silicosis fibrosis in silicon dioxide-induced RLE-6TN cells. miR-204-3p plays a negative regulatory role in silicosis fibrosis.
