| Silicosis is a systemic disease characterized by irreversible lung fibrosis,and it is one of the most serious occupational diseases in China,which is caused by long-term excessive inhalation of high concentration productive free silica dust.In the early clinical stage,the patients have slight or no obvious symptoms,but with the development of the disease,they may have cough,expectoration,hemoptysis,chest pain,chest tightness and other clinical manifestations.The basic pathological changes of silicosis are the formation of nodules and pulmonary interstitial fibrosis.Various studies have identified the pathological mechanisms of silicosis may include epithelial metaplasia,apoptosis,fibrocyte recruitment,fibroblasts proliferation,excessive deposition of extracellular matrix and differentiation of myofibroblasts.Due to the unclear pathogenesis of silicosis,there is no specific treatment.While with the development of science and deepening of research,emerging evidence has indicated that microRNAs(miRNAs,miRs)and EMT(epithelial-mesenchymal transition)play a crucial role in the process of the pathogenesis of fibrotic diseases.EMT is a cellular process that polar adjacent epithelial cells transform to non-polar mesenchymal cells which lack of cell-cell contact and increased cell mobility.With the stimulation of fibrotic factors,the alveolar type-Ⅱ-epithelial cells(AT2)will transform into fibroblasts or myofibroblasts via the process of EMT,thus promoting the development of pulmonary fibrosis.In the process of EMT,epithelial cells will gradually lose epithelial phenotype(decreased expression of epithelial markers such as E-cadherin and ZO-1),and gradually acquire the phenotype of mesenchymal cells(increased expression of mesenchymal markers such as a-SMA and vimentin).miRNAs are a class of 18～22-nucleotide-length small non-coding RNAs,which regulate the genes expression by pairing to the 3’UTR of the genes,thus repressing translation or degrading target mRNAs.Accumulating evidence indicates that miRNAs could regulate the process of pulmonary fibrosis via targeting fibrosis-related or EMT-related genes.Long non-coding RNAs(lncRNAs)are non-protein coding transcripts longer than-200 nucleotides and have drawn much attention recently.Emerging evidence has confirmed that several lncRNAs play pivotal roles in the regulation of various biological processes,such as cell proliferation and differentiation,metastasis and EMT.And the most acknowledged molecular mechanism of lncRNAs is served as"sponges" of miRNAs,modulating the activity of miRNAs.Therefore,IncRNAs are also known as competing endogenous RNAs(ceRNAs).Existing research shows that IncRNAs can regulate EMT in the mouse model of pulmonary fibrosis via regulating the expression of miRNAs.The purpose of our study was to explore the machnism of miR-503 in relieving silica-induced pulmonary fibrosis by establishing the in vivo murine silicosis model and in vitro HBE and A549 cells model.Objective:The silica-induced murine pulmonary fibrosis in vivo model,silica treated HBE and A549 cells in vitro model were established,and we also overexpressed miR-503 both in vivo and in vitro to discuss the role of miR-503 in relieving silicosis,which would provide theoretical data for the treatment of silicosis and other fibrotic diseases by using miRNAs or nucleic acid.Methods:(1)We formulated the silica-induced mouse pulmonary fibrosis model by intratracheal instillation of 50μl 50g/l silica turbid liquids for the three experimental group(7d,14d,28d)or 50μl physiological saline for the NS group,and without any treatment for the blank control group(n=10 for each group).And we observed the pathological changes of mouse lung tissues by pathological sections stained with hematoxylin and eosin.(2)We established silica-treated cell in vitro models by treating Human bronchial epithelial cells(HBE)and human lung adenocarcinoma A549 cells with silica suspension(prepared by mixing silica with MEM or 1640 median),and observed cell morphological changes through a microscope.(3)We detected the expression levels of miR-503 in different models by using qRT-PCR methods,and assessed the protein expression changes of EMT markers(epithelial marker:E-cadherin,mesenchymal markers:vimentin and a-SMA)in different models by using Western blot methods.We observed the changes of the fluorescence of a-SMA in silica-treated HBE cells by the method of immunofluorescence assay.(4)After silica installation,we respectively used miR-503 agomir or miR-503 mimic to up-regulate the expression of miR-503 in vivo and in vitro to construct the miR-503 intervention model.We then observed therapeutic effects of miR-503 in lung tissues by pathological sections stained with hematoxylin and eosin,the expression levels of miR-503 in the lung tissues and cells were detected by qRT-PCR methods,and the protein changes of EMT markers in the lung tissues and cells were determined by Western blot method.(5)We chose PI3K p85 which is related to EMT as the target gene of miR-503 predicted by some bioinformatics software combined with literature reading,and confirmed the combination through dual luciferase reporter gene experiments.After knocked down PI3K p85 the variation of protein expression of the downstream Akt,Snail and EMT markers were detected by using Western blot methods.(6)We performed the rescue assay by co-transfected pcDNA3.1-PI3K p85 plasmid with miR-503 mimic to further confirm the regulating relationship between miR-503 and PI3K p85.And then we detected the protein expression of PI3K p85,Akt,Snail and EMT markers via Western blot methods.(7)We predicted that lncRNA MALAT1 may target miR-503 through bioinformatics software and confirmed the combination by using RNA pull-down assay and dual luciferase reporter gene experiments.We knocked down the expression of lncRNA MALAT1 by using siRNA in two cell lines,and detected the changes of miR-503 by qRT-PCR methods and protein expression of PI3K p85,Akt,Snail and EMT markers ’by Western blot.Results1.The expression of miR-503 is down-regulated in the lung tissues of mice with silica-induced pulmonary fibrosis.The pathological changes displayed increased severity of lung fibrosis over time.On day 14 after silica instillation,we observed small silicon nodules from the pathological sections by hematoxylin and eosin(H&E)staining analysis.And on day 28,the typical fibrotic nodules changes were observed.Western blot analysis revealed that the E-cadherin protein level was decreased and the vimentin,a-SMA protein levels were increased along with the extension of the treatment time.To validate the expression level of miR-503 in this re-established model,qRT-PCR analysis was performed and displayed markedly decreased expression of miR-503,at most about five-fold of decrease in the day 28 group as compared with the control group.2.Increased miR-503 attenuates the EMT in vivo.The pathological analysis indicated that miR-503 agomir effectively relieves the severity and distribution of lung lesions compared with the silica plus miR-NC group.Consistently,the up-regulation of miR-503 increased the protein expression level of E-cadherin and decreased the expression levels of vimentin and a-SMA,thus alleviating the process of EMT.These results indicated that miR-503 alleviates the development and pathological process of mouse pulmonary fibrosis in vivo via the EMT-suppressive effects.3-miR-503 blocks the process of EMT via targeting PI3K p85.TargetScan bioinformatics software has predicted that PI3K p85 might be a functional potential target of miR-503,and some studies have reported PI3K p85 is an EMT-related gene.The results of dual luciferase assay indicated that miR-503 mimic could only down-regulate the relative luciferase activity of the wild type PI3K p85 reporter plasmid.The results of Western blot analysis revealed the expression of PI3K p85 was negatively correlated with the expression of miR-503.Knockdown of PI3K p85 by siRNA resulted in decreased vimentin and a-SMA expression,and increased E-cadherin expression,thus reversing the process of EMT.By performing the rescue assay,we found that overexpression of PI3K p85 largely counteracted the inhibitory effects of miR-503 mimic.Taken together,our results indicated that miR-503 alleviates the process of EMT by down-regulating the expression of PI3K p85.4.miR-503 influences EMT through PI3K/Akt/Snail signaling pathway.We further explored the downstream molecular signaling mechanisms that influence the process of EMT.Akt and Snail are two molecules downstream to PI3K,which were reported to modulate the expression of EMT markers and induce EMT.Therefore,we examined whether miR-503 could inhibit EMT through PI3K/Akt/Snail signaling pathway.Western analysis of the silica-induced mouse lung fibrosis tissues revealed that the variation trend of p-Akt and Snail protein expression was similar with PI3K p85.Consistently,in HBE cells,the protein expression levels of p-Akt and Snail were gradually increased while treated with different concentrations of silica for 48h and 200μg/ml silica for different time points.On the contrary,overexpression of miR-503 repressed the protein expression of p-Akt and Snail in vivo and in vitro.Silencing the expression of PI3K p85 also significantly attenuated the protein expression levels of downstream p-Akt and Snail.Our rescue experiment also showed that co-transfection with pcDNA3.1-PI3K p85 and miR-503 mimic restored the protein expression levels of p-Akt and Snail which were inhibited by miR-503 mimic.5.LncRNA MALAT1 promotes EMT via binding to miR-503 directly.We further explored the factors that result in the decrease of the expression of miR-503.We found a putative complementary sequence for miR-503 in IncRNA MALAT1 at position 6623-6650 via bioinformatics software.To further investigate whether lncRNA MALAT1 is a functional target of miR-503,the relative expression of IncRNA MALAT1 in silica-treated HBE cells was detected by qRT-PCR.The results showed that the expression of IncRNA MALAT1 is significantly up-regulated in the silica-treated group compared to the control group,which is negatively correlated with miR-503 expression.RNA pull-down and dual luciferase reporter gene assays were performed to identify the binding of miR-503 and IncRNA MALAT1.qRT-PCR analysis revealed that knockdown of lncRNA MAL ATI with siRNA could increase the expression level of miR-503.Western blot analysis also revealed that knockdown of lncRNA MAL AT1 together with the treatment of silica could obviously attenuate the process of EMT and restrain the expression of PI3K p85,p-Akt and Snail in HBE and A549 cells.These data suggest that lncRNA MALAT1 could affect the process of EMT in silica-induced pulmonary fibrosis via miR-503-PI3K/Akt/Snail signaling pathway.Conclusion:All these results suggest that the mechanism of miR-503 in inhibiting silica-induced pulmonary fibrosis is that miR-503 targets PI3K p85,inhibiting the signal transduction of downstream Akt/Snail signaling pathway thus alleviating epithelial-mesenchymal transition in pulmonary fibrosis.Moreover,we also found that IncRNA MALAT1 could act as the molecular "sponge" of miR-503 thus decreasing the expression of miR-503.When miR-503 is silenced,PI3K p85 bound to miR-503 will be released and thereby activate the downstream molecules,thus causing intensified the process of EMT. |
PDF Full Text Request