| Silicosis is a kind of systemic disease which is characterized by diffuse lung fibrosis induced by inhalation of dust with high concentration of free crystalline silica (SiO2). Morbility of silicosis was high in many industries such as coal mine. Silicosis is one of the most serious occupational diseases in China. It is characterized by the excessive proliferation of fibroblasts, differentiation of myofibroblasts and the excess deposition of extracellular matrix (ECM). The pathogenesis of silicosis has not been elucidated completely and there is no effective treatment hitherto. Therefore, more effective strategies for prevention, diagnosis and therapy of silicosis need to be explored. Recently, growing evidence has demonstrated that pulmonary fibrosis involve various cellular processes, including autophagy and microRNAs (miRNAs). Autophagy is a classic cellular pathway regulating the energy metabolism and self-renew of cell, and it plays crucial role in development and general homeostasis. In this pathway, cytosolic cargos including damaged organells and proteins are sequestered into autophagosomes, which are delivered to the lysosomes where they are enzymatically degraded and then recycled as metabolic precursors. Autophagy supplies raw material for the reconstruction, regeneration and restoration for cells, and regulates material circulation and reuse in cytoplasm. Recently, increasing evidence suggests that autophagy can impact either the pathogenesis or the progression of human diseases. And accumulating evidence indicated that miRNAs play important roles in modulating autophagic activity by targeting key autophagy proteins. miRNAs are 18-22 nucleotide-long noncoding RNAs that control biological events by either triggering degradation of their target mRNAs, and/or through inhibition of their translation by recognizing specific sequences generally present in the 3’UTR of target mRNAs. A number of published research showed that some miRNAs regulateautophagy by inhibiting the expression of key gene of autophagy, and impact the genesis and development of several human diseases such as cancer and neurodegenerative disease.Objective:Silicosis of mice was induced by silica suspension. We investigated the time-dependent changes of autophagic activity and miR-449a expression of lung tissues in mice, then verified in vitro by treating fibrobiasts with transforming growth factor (TGF)-β1. In addition, agomiR-449a was used to overexpress miR-449a in animal models. We tried to demonstrate the change of autophagic activity and miR-449a expression in vivo and vitro. Combined with relative literatures and the bioinformatics websites, we explored the regulatory relationship between miR-449a and Bcl2 in both lung tissues and cells, and further verified in vitro, so as to provide new clues to prevention, diagnosis and therapies for silicosis.Methods:(1) We formulated the silicosis animal model by exposing siliaca turbid liquid to mice via disposable trachea way. The dynamic changed process of lung fibrosis process in different time points were observed by pathological sections with hematoxylin-eosin stained. (2) The validations of miR-449a were done by quantitative reverse transcription-PCR (qRT-PCR) in different groups. And the markers of autophagy including p62 and LC3 were detected by Western Blot. (3) The fibroblasts were treated with TGF-β1, then assessed the expression of miR-449a using qRT-PCR and detected the expression of p62 and LC3 by Western blot. (4) C57BL/C mice were co-treated with agomiR-449a and SiO2, and the potential therapeutic significance of miR-449a was observed by pathological sections with hematoxylin-eosin stained. The validations of miR-449a were done by quantitative reverse transcription-PCR (qRT-PCR) in different groups (n= 5 per group). And the markers of autophagy including p62 and LC3 were detected by Western blot. NIH-3T3 and MRC-5 cells were transfected with miR-449a mimic, then treated with TGF-β1, and p62 and LC3 were detected by Western Blot. Immunofluorescence assays were used to detect the expression of p62. NIH-3T3 cells were co-transfected with miR-449a and GFP-LC3 plasmid to detect the GFP-LC3 dot formantion under a confocal laser scanning microscopy. Transmission electron microscope was used to observe the formation of autophagosome in NIH-3T3 cells. (5) The potential targeted genes were identified by using miRDB, TargetScan, PicTar and DIANA-microT which were available online. The Western Blot assays were used to test the expression of Bcl2, and the interaction of miR-449a and Bcl2 was determined by luciferase assays. (6) The NIH-3T3 cells were treated with TGF-β1, and Bcl2, p-ERK1/2 and ERK1/2 were tested by Western Blot assays.Results:1. Decreased activity of autophagy and downregulation of miR-449a were detected during the silica induced pulmonary fibrosis in a mouse model. Histological evaluation showed worse fibrosis after silica-induced lung injury in mice. The mice of the silica group showed a marked increase in the severity and the distribution of the lesions as compared to the control and saline groups. The distinct silicosis nodules were formed after exposure to silica 28 days. QRT-PCR assays were performed to analyze the endogenous expression of miR-449a and the results indicated that miR-449a was significantly down-regulated by treatment with silica in lung tissues of pulmonary fibrosis model. To investigate the pro-fibrotic efficacy of silica, the fibrotic markers vimentin, fibronectin and epithelial marker E-Cadherin were examined by immunoblot assay. We found that silica stimulated the expression of Collagen I, a-SMA, vimentin and fibronectin but suppressed the expression of E-Cadherin in a time-dependent manner. Furthermore, silica treatment significantly increase the p62 expression compared to the control group. And lipid conjugation of free LC3-I to the autophagic membrane-associated LC3-II was attenuated in the lungs after silica treatment.2. TGF-β1 simultaneously inhibited autophagy and miR-449a expression of fibroblast in vitro.Recombinant TGF-β1as used to treat the fibroblast cell lines (NIH-3T3 and MRC-5 cell line). The data showed that TGF-β1 could induce the synthesis of vimentin and fibronectin in NIH-3T3 and MRC-5 cells in a dose-dependent manner. We also tested varying time points up to 48 hours and found the same effects. We next checked the levels of p62 and LC3-Ⅰ/Ⅱ protein in NIH-3T3 and MRC-5 cells. The degradation of the autophagy receptor p62 following treated with TGF-β1 was inhibited. In line with these results, LC3-I/LC3-II conversions were attenuated when treated with TGF-β1. The expression of miR-449a in NIH-3T3 and MRC-5 cell lines treated with 2ng/ml TGF-β1 for 48 hours were detected by qRT-PCR. Compared to the non-treated cells, both fibroblast cell lines showed significant down-regulation of miR-449a.3. MiR-449a has anti-fibrotic effect in silica-induce lung fibrosis. The mice that were treated with miR-449a exhibited decreased lung fibrosis as determined by histological analyses of the lungs. Both the severity and the distribution of the lung lesions decreased following miR-449a administration compared with the silica group. MiR-449a overexpression also reduced Collagen I, a-SMA, fibronectin and vimentin expression in the fibrotic lungs. The expression of p62 was downregulated and the relative expression of LC3-II/LC3-I was upregulated after miR-449a treated.4. MiR-449a up-regulated autophagic activity in vitro. As hypothesized, miR-449a overexpression could attenuate TGF-β1-induced fibroblasts proliferation. Immunoblot assays indicated that Collagen I, α-SMA, fibronectin and vimentin were down-regulated by miR-449a. In line with these fibrosis-related markers, the levels of autophagy receptor p62 protein were less in miR-449a-transfected cell extracts. In accordance with the results of immunoblot assays, immunofluorescence microscopy analysis also showed diminished fluorescence for p62 upon miR-449a mimic transfection to TGF-β1 treated fibroblasts. And the TGF-β1-inhibited lipid conjugation of free LC3-I to the autophagic membrane-associated LC3-II was up-regulated in the extracts of cells following miR-449a mimic transfection. Subsequent GFP-LC3 tests in NIH-3T3 cell lines confirmed our data of immunoblot assays. Transmission electron microscopy analysis showed that autophagosomes in the cells treated with TGF-β1 was less than that of non-treated cells.5. Autophagy-related target of miR-449a.Bcl2 was identified as a miR-449a target by three bioinformatics tools. To confirm the bioinformatics-based predictions, we performed immunoblot analysis in control mimic or miR-449a mimic transfected cell extracts using a Bcl2-specific antibody. Indeed, Bcl2 protein levels were decreased in NIH-3T3 cells which were overexpressing miR-449a. Co-transfection of miR-449a together with the wild-type luciferase vector in NIH-3T3 cells showed a significant decrease in the luciferase activity compared with the group co-transfected with control mimic and wild-type luciferase vector. In contrast, miR-449a had no significant effect on the levels of luciferase activity when transfected with mutant construct.6. TGF-β1 interrupted the degradation of Bcl2 through activation of Erkl/2.The expression of Bcl2 in NIH-3T3 and MRC-5 cells were enhanced when treated with TGF-β1. To investigate the role of ERK activation in the induction of Bcl2 by TGF-β1, we measured the expression of phosho-ERK1/2 (p-ERK1/2), the activated forms of ERK1/2 proteins, by western blot analysis. TGF-β1 induced an increased expression of p-ERK1/2 in a dose-and time-dependent manner.Conclusion:In the progression of pulmonary fibrosis induced by SiO2, miR-449a expression was downregulated and the activity of autophagy was inhibited in fibrotic lung tissues. Overexpression of miR-449a attenuated silica-induced lung fibrosis and TGF-β1-induced fibroblast differentiation. MiR-449a regulates the activity of fibroblasta through regulation of the expression of Bcl2, a crucial mediator of autophagy. Autophagy may inhibit fibrogenesis by regulating the proliferation of fibroblasts. TGF-β1 may increase the expression of BcI2 via MAPK/ERK pathway. These results suggest that miR-449a increase the autophagic activity of lung fibroblast by targeting Bcl2 and inhibit the progression of lung fibrosis. |
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