The Anti-fibrotic Effects And Mechanisms Of MiRNA-486-5p In Pulmonary Fibrosis

microRNAs (miRNAs) are short endogenous noncoding RNAs which play pivotal roles in a broad range of biological processes including regulation of the intracellular signaling pathways involved in diseases. Since the identification of the first miRNA, approximately more than two thousands and five hundreds miRNAs have been characterized in the human setting (miRbase v21) (http://www.mirbase.org/). MiRNAs promote mRNA degradation or inhibit translation by binding to complementary sequences in mRNA 3’untranslated regions (UTRs). MiRNAs play an important role in lung cells, where they have been implicated in cell proliferation, apoptosis, differentiation, etiology and progression of fibrotic diseases. Alteration of specific miRNAs expression profiles can produce dramatic effects on fibrosis, suggesting that rational modulation of miRNA expression can be regarded as a novel approach to develop innovative therapeutic strategies. Toward this, we sought to investigate whether the study of miRNAs in the context of pulmonary fibrosis could inform new perspectives on mechanisms, as well as diagnostic and therapeutic opportunities. We performed genome-wide profiling of miRNA expression in lung tissues of silica-induced mouse pulmonary fibrosis by microarray. Seventeen miRNAs were selected to be validated by qRT-PCR according to the fold changes between silica and control groups at each time point. Five miRNAs, miR-21, miR-455, miR-151-3p, miR-486-5p and miR-3107, were identified to consistent with microarray results which also confirmed in bleomycin-induced mouse lung fibrosis. Then, miR-486-5p was selected for the further study. The anti-fibrotic role and possible mechanisms of miR-486-5p in pulmonary fibrosis were domenstrated in our present study. Besides, we explored whether miR-486-5p could be a potential therapeutic target for pulmonary fibrosis.Objective(1) To identify miRNAs related with silicosis via analyzing the genome-wide profiling of miRNA expression in lung tissues of silica-induced mice pulmonary fibrosis and validating the microarry results; (2) To verify the anti-fibrotic role of miR-486-5p in silica-and bleomycin-induced pulmonary fibrosis via over-expressing miR-486-5p by ago-miR-486-5p; (3) To elucidate the mechanisms for the anti-fibrotic role of miR-486-5p in pulmonary fibrosis in vitro. Above all, the aim of our present study was to provide new clues for potential therapeutic targets of pulmonary fibrosis.MethodsPulmonary fibrosis models were conducted intratrachally when mice were exposed to silica or bleomycin. Then microRNA microarray was performed in the lung tissues harvested from the silica-induced pulmonary fibrosis model. One mouse at each time point was selected on the basis of H&E staining sections. Quantity real time polymerized chain reaction was used to validate the result of microarry in silica-and bleomycin-induced pulmonary fibrosis models. miR-486-5p expression in serum samples and lung tissues of patients with silicosis was measured by qRT-PCR as well as in lung tissues of patients with idiopathic pulmonary fibrosis. AgomiR-486-5p was used to interfere the pulmonary fibrosis that induced by silica or bleomycin and H&E staining, Masson’s staining, immunohistochemistry as well as fibrosis scores were performed to evaluate the effect of miR-486-5p on pulmonary fibrosis. qRT-PCR and Western blot assays were used for detecting whether miR-486-5p could affect the fibrotic activity of fibroblasts. Bioinformatics software and dual luciferase reporter gene assays were used to explore the target genes of miR-486-5p. CCK-8 and flow cytometry assays were performed to illustrate whether miR-486-5p could interfere with TGF-β1-induced proliferation in fibroblast cells.Results1. Screening and validating the ectopic miRNAs in silica-induced pulmonary fibrosisPulmonary fibrosis models were conducted successfully when mice were exposed to silica or bleomycin intratracheally. miRNA microarray was performed with total RNA isolated from mice lung tissues which were harvested on days 0,3,7,14,28 and 56 after silica challenge. Seventeen miRNAs were selected to be validated by qRT-PCR according to the fold changes between silica and control groups. Five miRNAs were identified to be consistent with microarray results which was also confirmed in bleomycin-induced mouse lung fibrosis, including up-regulated miR-21 as well as down-regulated miR-455, miR-151-3p, miR-486-5p and miR-3107.2. Enhancement of miR-486-5p expression attenuates experimental pulmonary fibrosismiR-486-5p was chosed for the further study and miR-486-5p levels were found to be decreased in the serum of patients with silicosis as well as in lung tissues from cases with silicosis and IPF. Then, we hypothesized that over-expression of miR-486-5p could relieve pulmonary fibrosis and cholesterol-modified miR-486-5p mimics (agomiR-486-5p) were used to restore the expression of miR-486-5p in silica-and bleomycin-induced pulmonary fibrosis models. We found that enhancement of miR-486-5p attenuated lung fibrosis induced by silica and bleomycin as assessed by histological analyses of the lungs and Masson’s trichrome assays for collagen deposition. Over-expression of miR-486-5p also reduced a-SMA expression in fibrotic lungs, suggesting that miR-486-5p could diminishe myofibroblast differentiation. These results indicates that enhancement of miR-486-5p expression could attenuate pulmonary fibrosis in vivo.3. The possible mechanisms for the anti-fibrotic role of miR-486-5pBioinformatics softwares were used to predict that SMAD2 and Col6a6 might be the potential target of miR-486-5p and dual luciferase reporter gene assays suggested that transfection of miR-486-5p significantly reduced the normalized luciferase activity for the reporter containing wild type, but not mutant type with mutations in the predicted miR-486-5p site, validating that miR-486-5p could regulate SMAD2 and Col6a6 directly.To verify whether miR-486-5p could be involved in the process of pulmonary fibrosis via TGF-β1 signal pathway, we observed the expression of miR-486-5p in fibroblasts (NIH-3T3 cells) treated with TGF-β1. Then, we found that treatment of mouse fibroblasts with TGF-β1 resulted in decreased levels of miR-486-5p and increased mRNA expression of the myofibroblast differentiation markers Fn and a-SMA. Next, we addressed whether miR-486-5p regulates fibrogenesis in mouse fibroblasts. NIH-3T3 cells were cultured in presence of TGF-β1 for 24 hours after transfection with miR-486-5p mimics or control mimics (miR-NC). We found that over-expression of miR-486-5p levels in fibroblasts decreased p-SMAD2 protein levels as well as a-SMA, Fn and CTGF expression at both protein and mRNA levels, compared to those in cells with control mimics. However, when miR-486-5p was knocked down with miR-486-5p inhibitors in mouse fibroblast cells, we found that the p-SMAD2 protein expression increased in anti-miR-486-5p-transfected cells. Meanwhile, TGF-β1-induced expressions of a-SMA, Fn and CTGF were further enhanced in anti-miR-486-5p-transfected cells, compared to those in cells with control inhibitors. These data suggest that miR-486-5p negatively regulates the proliferation and fibrogenic activity of fibroblasts. Decreasing miR-486-5p expression could relieve the inhibition on SMAD2 expression, contributing to strengthening the TGF-β1 signal pathway. Furthermore, the proliferation and fibrogenic activity in fibroblasts were improved by the reduction of miR-486-5p. These results indicate that miR-486-5p would be involved in the process of pulmonary fibrosis via regulating TGF-β1 signal pathway.Finally, we sought to illustrate whether miR-486-5p could interfere with TGF-β1-induced proliferation in fibroblasts cells. CCK-8 assay and flow cytometry assay were performed to explore the effects of miR-486-5p on proliferation of fibroblasts. CCK-8 assay showed significant increases in cell number in the TGF-β1 treatment group, compared to the control group. However, decreased cell numbers were seen in groups with miR-486-5p mimics whereas not in the presence of miR-NC transfection, indicating that miR-486-5p successfully suppressed TGF-β1-induced proliferation of fibroblasts. Then we examined the effects of miR-486-5p on cell-cycle progression in NIH-3T3 cells and observed a significant increase in S phase and a decrease in G1 phase after TGF-β1 treatment, whereas, these effects could be restored by miR-486-5p, supporting a role for miR-486-5p in repressing TGF-β1-induced proliferation through effects on rising S phase and decreasing cell-cycle arrest. These results indicated that miR-486-5p could regulate the TGF-β1 signal pathway, contributing to affecting the proliferation and activity of fibroblasts.ConclusionsIn the present study, we delineated a specific miRNA profile associated with silica-induced pulmonary fibrosis and confirmed the role of dysregulated miRNAs in the pathogenesis of silica-and bleomycin-induced lung fibrosis. The following miRNAs, such as miR-21, miR-455, miR-151-3p, miR-486-5p and miR-3107, were validated to be differentially expressed in fibrotic lung tissues of mice. Compared to normal lung tissues, miR-21 was up-regulated in fibrotic lung tissues, whereas miR-455, miR-151-3p, miR-486-5p and miR-3107 were down-regulated. Additionally, compared with healthy donors, miR-486-5p was found to be decreased in serum of silicosis patients, as well as in lung tissues of silicosis and IPF cases. Consequently, miR-486-5p may represent a primary pathogenic mechanism in the development of lung fibrosis. We found that enhancement of miR-486-5p expression could attenuate pulmonary fibrosis in vivo. Besides, we illustrated that miR-486-5p might be involved in the process of pulmonary fibrosis via TGF-β1 signal pathway in vitro. Our dominant finding was that over-expression of miR-486-5p could alleviate pulmonary fibrosis induced by silica and bleomycin, suggesting that miR-486-5p could be a potential therapeutic target for fibrotic diseases.