MiR-448-5p Inhibits TGF-?1-induced Epithelial-Mesenchymal Transition Of Bronchial Epithelial Cells By Regulating Six1 In Asthma

Asthma is one of the common chronic respiratory diseases,its morbidity has increased on a global scale,and the asthma has become a severe public health problem.The frequent occurrence of injury and repair initiated by chronic inflammation could lead to structural changes in the airway,collectively termed airway remodeling.Airway remodeling is characterized by airway wall thickening,subepithelial fibrosis,increased smooth muscle mass,angiogenesis and increased mucous glands.Although the effective implementation of Global initiative for asthma(GINA)program has achieved some clinical effects,the main drugs of this program,such as glucocorticoids,leukotrienes antagonists,are effective in reducing airway inflammation and alleviating asthma symptoms,but the effect on airway remodeling of asthma is weak.Therefore,it is necessary to explore the molecular mechanism of airway remodeling from gene,protein,cell and other levels,and explore the key target and molecular mechanism of effective control of airway remodeling in asthma,so as to lay the foundation for the development of drugs that improve airway structure or reverse airway remodeling.Epithelial-mesenchymal transition(EMT)is an orchestrated series of events,in which differentiated epithelial cells undergo a phenotypic transition to mesenchymal cells,often fibroblasts and myofibroblasts.During EMT,the epithelial cells lose intracellular junctions,leading to dissociation from the surrounding cells,acquire mesenchymal-like characteristics and become able to migrate away from the original location.The expression of ZO-1 and E-cadherin were decreased in epithelial cells while vimentin and a-SMA were increased.The phenotype transformation of EMT can be divided into three types:Type 1,which is mainly related to embryo formation,embryo development,neural crest development and gastrulation.Type 2.which is mainly related to wound healing and tissue regeneration and repair.Type 3.which is mainly related to the migration and invasion of malignant tumor cells.At present,it has been found that EMT can be regulated by many signaling pathways,among which TGF-P signaling pathway is the major avenue of approach.TGF-? receptor can be divided into three types.Type ? and type ? receptors with transmembrane structure play a role in signal transduction.TGF-? receptor has intrinsic serine/threonine kinase activity and induces specific signal transduction cascade reaction.TGF-?1 first binds to the type ? receptor on the cell surface,and the serine/threonine kinase domain in the activated type ? receptor cytoplasmic domain promotes the phosphorylation of GS domain in the type ? receptor cytoplasmic domain,which makes the type ?receptor activated and leads to initiating the signal transduction in the cell.Clinical and in vitro experiments showed that TGF-?1 plays an important role in airway remodeling and EMT transformation of asthma.It has been reported that TGF-?1 can play a role through various signaling pathways,such as Smad signaling pathway,phosphatidyl inositol-3-hydroxykinase/protein kinase(P13K/Akt)pathway,and upregulate the expression of transcription factors such as twist,snail,ZEB1.slug,ZEB2,so as to regulate the stability of cytoskeleton protein and intercellular connection.Our previous studies have found that the expression of TGF-?1 and Smad3 were upregulated in the lung tissue of asthmatic mice.This indicating that the TGF-?1/Smad3 signal pathway may be related to the EMT transformation in asthma.MicroRNAs(miRNAs)are a kind of short non coding single chain RNAs,which are composed of 18-25 nucleotides.They are highly conserved and tissue-specific in the process of biological evolution and are the basic regulators of gene expression.By binding to target gene 3'-untranslated regions(3'-UTR),miRNAs can induce specific mRNA degradation or inhibit translation,thus regulating the transcription and expression of specific genes.MiRNAs can participate in many biological processes,such as cell proliferation,differentiation and stress response.The relationship between gene expression and miRNAs is complex.A miRNA can regulate multiple genes,and a single gene can also be regulated by the same multiple miRNAs.MiRNAs and target genes form a network regulation mode in a variety of diseases.It was found that miRNAs can participate in the development of asthma.Some studies have found that miR-378 expression in blood and lung tissue of children with asthma is significantly higher than that of healthy children.In vitro experiments show that miR-378 may promote the proliferation of smooth muscle cells,increase their anti-apoptosis ability,and promote airway remodeling through RAS,MAPK or Ca2+ signal pathway.Another study showed that miR-323-3p may inhibit the production of IL-22 by T cells through downregulating TGF-? signal pathway,thus blocking the development of asthma.Cheng D et al found that miR-448 can reduce the metastasis and invasion of NSCLC by inhibiting EMT transformation.It has been found that downregulating the expression of miR-448 and upregulating the activity of NF-?B signaling pathway lead to EMT transformation and tumor development.However,the underlying role of miR-448-5p in the development of asthma has not been explored.The online database Target Scan 6.2 indicated that Sine oculis homeobox homolog 1(Six1)is a binding target of miR-448-5p,suggesting that Sixl may be the target gene of miR-448-5p.Sixl gene is a newly discovered homeobox gene,which is located on human chromosome 14q23 and encodes a protein composed of 183 amino acids.The specificity of the target gene is determined by the specificity of its binding to DNA,which is necessary for the development of brain,ear,eye,muscle and kidney.As a kind of transcription regulatory factors with specific spatiotemporal expression patterns,Sixl can specifically regulate the expression of target genes and play an important role in cell growth,differentiation,proliferation and apoptosis.Six1 was initially identified as a TGF-?-inducible gene and has been cloned in mice and humans.The mechanism by which Sixl influences signaling events in target cells has been the focus of several studies that have identified at least five potential Six1 signaling pathways,including influences of Six1 on the bone morphogenic protein pathway to inhibit phosphorylated Smad 1,3,8 signaling,on the AKT pathway,on the AMP-activated protein kinase pathway,on the TLR4/CD14 pathway,and on the Na/K-ATPase membrane potential.Although the role of Sixl is emerging in the process of disease,its specific role and regulatory mechanism in asthma are rarely reported.In this study,we provide experimental evidence that miR-448-5p is essential for EMT and pulmonary fibrosis in asthmatic mice.Moreover,we reveal that a novel post-transcriptional regulatory mechanism of Six1 expression is mediated by miR-448-5p.These results may enhance our understanding of the molecular mechanisms underlying the pathogenesis of peribronchial fibrosis,which may provide opportunities to prevent and treat asthma effectively.Part I.The construction of epithelial-mesenchymal transition model of humanbronchial epithelial cells induced by TGF-?1ObjectiveEMT is an important mechanism of airway remodeling and fibrosis in asthma.In this part,we construct an EMT transformation model used TGF-?1 in human bronchial epithelial cells,which can provide a model for the following study.Methods1.TGF-?1 stimulates human bronchial epithelial cells 16HBE at different concentrations.2.The morphological changes of human bronchial epithelial cells stimulated by TGF-?1 were observed under microscope.3.Western blot was used to detect the expression of EMT markers in human bronchial epithelial cells.Results1.After TGF-?1(lOng/ml)intervention,human bronchial epithelial cells lost their original cobblestone shape and exhibited an elongated,spindle-shapedmorphology,characteristic of fibroblasts.2.After TGF-?1 intervention,the expression of E-cadherin in human bronchial epithelial cells was significantly lower than that in the control group(P<0.05),and the expression of a-SMA and Vimentin were significantly increased(P<0.05).3.After TGF-?1(1ng/ml?10ng/ml)treatment,the expression of fibronectin and collagen IV in human bronchial epithelial cells was significantly higher than that in the control group(P<0.05).4.In the EMT model of human bronchial epithelial cells,when the intervention concentration of TGF-?1 was 10ng/ml,the changes of EMT phenotype markers and fibrosis related proteins were dramatically obvious.ConclusionIn the present study,the data demonstrated that bronchial epithelial cells underwent a transition from an epithelial into fibroblast like with obvious changes in phenotypic markers following activation with TGF-?1(10ng/ml).The EMT model of human bronchial epithelial cells was successfully constructed for the following study.Part II.The effect of miR-448-5p on TGF-p1-induced epithelial-mesenchymal transition and airway fibrosis in human bronchial epithelial cellsObjectiveMiR-448-5p is considered to be a miRNA related to EMT transformation.This study aimed to analyze the expression of miR-448-5p in lung tissue of asthmatic mice and TGF-?1-induced EMT model,and explore the effect of miR-448-5p on TGF-?1-induced EMT and airway fibrosis and its molecular mechanism.Methods1.The expression of miR-448-5p in lung tissue of asthmatic mice and human bronchial epithelial cells induced by TGF-?1 was detected by RT-PCR.2.miR-448-5p mimic and miR-448-5p inhibitor were transfected into human bronchial epithelial cells,and TGF-?1 intervention was given.The expression of EMT related markers was detected by RT-PCR.3.miR-448-5p mimic and miR-448-5p inhibitor were transfected into human bronchial epithelial cells,and TGF-?1 was given.RT-PCR was used to detect the expression of fibronectin and collagen ?.4.miR-448-5p mimic and miR-448-5p inhibitor were transfected into human bronchial epithelial cells,and TGF-?1 intervention was given.Western blot was used to detect the expression of Smad3 in the cells and analyze the possible mechanism of miR-448-5p.Results1.RT-PCR showed that the expression of miR-448-5p was significantly reduced in the lung tissue of asthmatic mice and EMT transformation model(P<0.05).2.Up-regulation of miR-448-5p expression increase the expression of E-cadherin and decrease the expression of Vimentin in human bronchial epithelial cells(P<0.05).Down-regulation of miR-448-5p significantly reduce the expression of E-cadherin in human bronchial epithelial cells treated by TGF-?1,while the expression of Vimentin increased significantly(P<0.05).3.Up-regulation of miR-448-5p expression significantly reduce the expression of fibronectin and collagen ? induced by TGF-?1(P<0.05).Downregulation of miR-448-5p expression significantly increased the expression of fibronectin and collagen IV induced by TGF-?1(P<0.05).4.The expression of phosphorylated Smad3 in miR-448-5p mimic group was significantly lower than that in TGF-?1 group(P<0.05).The expression of phosphorylated Smad3 in miR-448-5p inhibitor group was significantly higher than that in TGF-?1 group(P<0.05).ConclusionmiR-448-5p can inhibit TGF-?1-induced EMT and airway fibrosis in human bronchial epithelial cells,and TGF-?/Smad3 signaling pathway may play an important role in this process.Part III.miR-448-5p inhibits TGF-?1-induced epithelial-mesenchymal transition of bronchial epithelial cells by regulating Six1 in asthmaObjectiveThe online database Target Scan 6.2 indicated that Sine oculis homeobox homolog 1(Sixl)is a binding target of miR-448-5p,suggesting that Sixl may be the target gene of miR-448-5p.In this part,we intend to reveal the target regulation relationship between miR-448-5p and Six1,and the regulatory effect of Six1 on TGF-?/Smad signal pathway,as well as the related molecular mechanism.Methods1.Western blot was used to detect the expression of Six1 in human bronchial epithelial cells after transfected with miR-448-5p mimic or miR-448-5p inhibitor.2.To construct the airway remodeling model of asthma and the EMT model of human bronchial epithelial cells induced by TGF-?1.The expression of Six1 in the lung tissue and human bronchial epithelial cells was detected by immunohistochemistry or Western blot.3.Six1 interference plasmid was transfected into human bronchial epithelial cells,and TGF-?1 was given to intervene.The expression of EMT markers and fibronectin were detected by Western blot.4.The Sixl expression plasmids and miR-448-5p mimic were co-transfected into human bronchial epithelial cells,and TGF-?1 was given.RT-PCR was used to detect the expression of EMT markers,fibronectin and collagen ? in human bronchial epithelial cells.5.Six1 interference plasmid was transfected into human bronchial epithelial cells,and TGF-7?1 was given to intervene.Western blot was used to detect the expression of Smad3 in human bronchial epithelial cell.Results1.Double luciferase showed that the activity of luciferase decreased significantly in the wild-type vector of Sixl 3'-UTR after co-transfection with miR-448-5p mimic compared with the control group,but the mutant vector did not change significantly.Western blot showed that the expression of Sixl in human bronchial epithelial cells after miR-448-5p mimic transfection was significantly lower than that in TGF-?1-induced group(P<0.05),while the expression of Sixl in human bronchial epithelial cells after miR-448-5p inhibitor transfection was significantly higher(P<0.05).2.Immunohistochemistry and Western blot showed that the expression of Six1 in lung tissue of asthmatic group was significantly higher than that of control group(P<0.05).After TGF-?1 intervention,the expression of Sixl in human bronchial epithelial cells was significantly higher than that in the control group(P<0.05).3.After transfection of Sixl interference plasmid into human bronchial epithelial cells,Western blot showed that the expression of E-cadherin was significantly higher than that of TGF-?1 alone(P<0.05),the expression of vimentin was significantly lower(P<0.05),and the expression of fibronectin was lower than that of TGF-?1 alone(P<0.05).4.RT-PCR showed that the expression of E-cadherin was significantly decreased in the group of transfected with Sixl plasmids and miR-448-5p mimic(P<0.05),the expression of vimentin was significantly increased(P<0.05).The expression of fibronectin and collagen ? ware significantly higher than that in transfected with miR-448-5p mimic alone group(P<0.05).5.After transfection of Sixl interference plasmid into human bronchial epithelial cells,the results showed that the expression of phosphorylated Smad3 was significantly decreased(P<0.05).ConclusionTaken together,our data demonstrated that miR-448-5p over-expression prevented TGF-?1-stimulated EMT in bronchial epithelial cells by targeting Sixl expression.The miR-448-5p/TGF-?1/Six1 link may play roles in the progression of EMT and pulmonary fibrosis in 16HBE cells and may represent novel targets for strategies for preventing airway remodeling in asthma.