The Anti-fibrotic Effects And Mechanisms Of MicroRNA-146a In Pulmonary Fibrosis

Background:How to treat pulmonary fibrosis is still a problem.Therefore,more in-depth study of its pathogenesis is needed to find more therapeutic targets. MicroRNA-146 a has a negative regulatory role in the NF-kappa B signaling pathway.Based on our team's previous studies,we found that the expression of microRNA-146 a was abnormal in patients with Sjogren's syndrome and pulmonary fibrosis.Combining other people's research we assume that microRNA-146 a may be involved in the occurrence and development of pulmonary fibrosis.In this study,we intend to investigate the role of microRNA-146 a in pulmonary fibrosis and its possible molecular mechanism by observing the expression of microRNA-146 a in pulmonary fibrosis model and cells.This dissertation consists of three parts:Part ? Expression of microRNA-146 in bleomycin-induced murine pulmonary fibrosis modelObjective:To investigage the changes of miR-146 a in thedevelopment of pulmonary fibrosis induced by bleomycin.Methods:C57BL/6wild-tybpe mices were randomly divided into two group:blelmycin group and saline group.Blemycin group: intratracheal instillation of bleomycin;saline group: intratracheal instillation of saline.Lung tissues were obtained at day 7,14,21 and 28.The pathological changes in lung tissue were observed after HE staining and Masson's staining.The collagen content in lung tissues were assessed after detecting hydroxyproline content.The protein expression of ?-SMA?TGF-?1?COL I and COL III were examined by immunohistochemical techniques.The expression of miR-146 a were examed by qRT-PCR.The protein of Smad4 were detected by western blot.Results:1)Compared with saline group,bleomycin group mice showed progressive pulmonary fibrosis and alveolitis 7-28 days after modeling.The content of hydroxyproline increased progressively.At the same time,the protein of alpha-SMA,TGF-beta 1,COL I and COL III increased in lung tissue.The change was most obvious on the 21 st day.2)The expression level of microRNA-146 a in lung tissue of BLM mice was lower than that of Saline mice,and the expression level decreased with the progress of fibrosis.3)The expressioin of Smad4 protein in lung tissue increased with the progress of fibrosis.Conclusion:The expressioin of miR-146 a was downregulated in bleomycin-induced murine pulmonary fibrosis model.Part ? Role of microRNA-146 a in phenotypic transformation of lung fibroblasts and its molecular mechanismObjective: To study the effects of overexpression and inhibition of miR-146 a on phenotypic transformation of mouse lung fibroblasts.Methods: To explore the appropriate concentration and time of stem-induced fibroblast transformation after intervention with TGF-beta 1 at different concentrations or at different time of action,the expression of microRNA-146 a in cells was detected at the same time.NIH/3T3 cells were divided into five groups.Except one group cell,the other four groups cell were interfered with miR-146 a mimic,mimic-negative,sponge and sponge-negative by lipid transfection.After fibroblasts were induced into myofibroblasts by TGF-beta 1,Cell proliferation was detected by CCK-8,apoptotic of myofibroblasts were investigated by flow cytometry methods.The protein expression of ?-SMA ? TGF-?1 ? COL I and COL III were examined by western blot.Direct Target Genes of miR-146 a was detected by bioinformatics Prediction Software and Double Luciferase Assay.Result:1)After Pulmonary fibroblasts induced by different concentrations of TGF-beta 1,the protein of ?-SMA ? TGF-?1 ? COL I and COL III were increased in group 10ug/ml and 20ug/ml.The expression of miR-146 a was decreased in group 1ug/ml,10ug/ml and 20ug/ml.When we intervented pulmonary fibroblasts with 10 ng/ml of TGF-beta 1,the level of miR-146 a decreased at day2 and day3.2)It was found that over-expression of microRNA-146 a could decrease theexpression of Smad4 protein.At the same time,the expression levels of a-SMA,collagen I and collagen III were also decreased.Inhibiting the expression of microRNA-146 a in lung fibroblasts can increase the expression level of Smad4 induced by TGF-beta 1,At the same time,the expression levels of a-SMA,type I collagen and type III collagen were increased.These results suggest that microRNA-146 a may regulate the progression of pulmonary fibrosis by interfering with the expression of Smad4.Compared with the control group,the proliferation ability of sponge fibroblasts increased.Apoptosis in the sponge group decreased significantly.These results suggest that inhibiting the expression of microRNA-146 a can increase cell proliferation by reducing apoptosis.3)Prediction and Confirmation of the Target Gene of miR-146aThe target genes were predicted by bioinformatics prediction software.Smad4 were found to be the target genes of microRNA-146 a.CONCLUSION: The expression level of microRNA-146 a can affect the phenotype transition of pulmonary fibroblasts through TGF-beta 1/Smad signal transduction.Part ? Effects of interfering with the expression of miR-146a on pulmonary fibrosis in model miceObjective: To explore the effect of over-expression of microRNA-146 a on bleomycin-induced pulmonary fibrosis in mice.Methods: Mice were divided into three groups: blank control group(saline),negative control group(mimic-NC),miR-146 a mimic group(mimic).When the mice were intratrachealed with blomycin,they were injected of miR-146 a mimic,miR-NC and saline through vena caudalis.At day21,the expression of miR-146 a were detected,and he pathological changes in lung tissue were observed after HE staining and Masson's staining.The collagen content in lung tissues were assessed after detecting hydroxyproline content.The protein expression of ?-SMA were examined by immunohistochemical techniques.The expression of miR-146 a were examed by qRT-PCR.Results:The exression of miR-146 a were higher in mimic group,that meaned the Intervention successed.We observed,in mimic group,pulmonary fibrosis and alveolitis was controled and the content of hydroxyproline decreased.The expression of alpha-SMA also decreased in mimic group.Conclusion: Overexpression of microRNA-146 A inhibits bleomycininduced pulmonary fibrosis.In summary,through the study of animal models of pulmonary fibrosis,we confirmed that microRNA-146 A is related to the occurrence and development of pulmonary fibrosis.Overexpression of miR-146 a can alleviate bleomycin-induced pulmonary fibrosis.It is preliminarily confirmed at cellular level that microRNA-146 a can participate in the process of pulmonary fibrosis through TGF-beta 1/Smad signaling pathway.This study provides an experimental basis for the targeting therapy of pulmonary fibrosis with microRNA-146 a in the future.