Mechanism of miR-21 targeting Smad7 in pulmonary fibrosis of A549 cells induced by beryllium sulfate

Faqiu QI; Xiaohui Chen; Hongya Liu; Feng Zhao; Youjuan FU; Suzhen Guan; Kai Wang

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Mechanism of miR-21 targeting Smad7 in pulmonary fibrosis of A549 cells induced by beryllium sulfate

VernacularTitle:硫酸铍诱导A549细胞纤维化中miR-21靶向调控Smad7的机制
Author: Faqiu QI ¹ ; Xiaohui CHEN ¹ ; Hongya LIU ¹ ; Feng ZHAO ¹ ; Youjuan FU ¹ ; Suzhen GUAN ¹ ; Kai WANG ¹
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1. School of Public Health and Management/Ningxia Key Laboratory of Environmental Factors and Chronic Disease Control, Ningxia Medical University, Yinchuan, Ningxia 750004, China.
Publication Type:Experiment
Keywords: microRNA-21; Smad7; beryllium sulfate; pulmonary fibrosis; transforming growth factor-β1
From: Journal of Environmental and Occupational Medicine 2022;39(2):206-211
CountryChina
Language:Chinese
Abstract: Background The pathogenesis of beryllium-induced pulmonary fibrosis is unknown and there is no specific treatment for the disease as yet. MicroRNA (miRNA) may play a role in the process of beryllium-induced pulmonary fibrosis. Objective To construct a microRNA-21 (miR-21) interfering cell line, and to investigate the effect of miR-21 on beryllium sulfate (BeSO₄)-induced fibrosis in human lung adenocarcinoma alveolar basal epithelial cells (A549 cells) and its potential mechanism. Methods The miR-21 target genes were predicted by the online database miRBase and verified by experiments using dual luciferase reporter gene. After transfecting A549 with miR-21interference lentivirus, puromycin was used to select a stable cell line. An in vitro model of pulmonary fibrosis was established using BeSO₄ infecting A549 cells with a concentration of 10 μmol·L⁻¹ and an exposure time of 48 h. Then the treated cells were divided into control group, model group, miR-21 interference group, and miR-21 interference control group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the relative expression level of miR-21 gene. Western blotting was used to detect the relative expression levels of TGF-β1/Smads pathway related proteins [Smad2, Smad3, p-Smad2, p-Smad3, Smad7, and transforming growth factor-β1 (TGF-β1)], myofibrosis cell marker α-smooth muscle actin (α-SMA), andextracellular matrix collagen-I (COL-I) and collagen-Ⅲ (COL-Ⅲ). Results The miRBase predicted that miR-21 had a binding site with Smad7, and the results of the dual luciferase reporter gene experiment showed that the target gene of miR-21 was Smad7. The construction of miR-21 interfered with A549 cell line was successful. Compared with the control group, the relative expression of miR-21 gene in the model group increased by 97.57%; the relative expression of Smad7 protein in the model group decreased by 15.48%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ increased by 13.55%, 35.72%, 18.35%, 35.75%, 25.52%, 31.58%, 24.61%, and 11.66% respectively (P<0.05). Compared with the interference control group, the miR-21 gene expression level in the interference group decreased by 28.96%; the relative expression of Smad7 protein increased by 19.07%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ decreased by 8.01%, 19.95%, 14.56%, 19.37%, 11.95%, 10.96%, 18.81%, and 31.36% repectively (P<0.05). There was no statistically significant difference in the gene abd protein expression levels of each gene between the model group and the interference control group (P>0.05). Conclusion In an in vitro model of pulmonary fibrosis induced by beryllium compounds, miR-21 may promote fibrosis by targeting Smad7 to regulate the TGF-β1/Smad signaling pathway.