Study On The Role And Mechanism Of MiR-221-5p Negatively Regulating MUC5B Gene Mediating EMT In Pulmonary Fibrosis

Background and purpose:Idiopathic pulmonary fibrosis（IPF）is a progressive fibrotic lung disease that affects 5 million people worldwide,with a median survival of 3-5 years^[1]and one of the worst prognosis subtypes of interstitial lung disease.The pathogenesis of IPF is still unclear.Currently,it is thought to be the result of multiple factors involved,among which the Epithelial-mesenchymal transition（EMT）theory plays an important role.The whole genome of 8 ILD patients from one family was sequenced,and the MUC5B rs2943512 mutation was found in the whole genome sequence of this family.Single nucleotide polymorphism（SNP）in the promoter region of the MUC5B gene may promote the expression of MUC5B in the lung and be one of the risk factors for familial interstitial lung disease.MUC5B is a major gel-forming mucin in the human airway^[2],which is mainly secreted by submucosal glands and is essential for normal lung defense and mucociliary clearance^[3].Most mucoobstructive diseases,such as cystic fibrosis and chronic obstructive pulmonary disease,are associated with airway mucus plugs composed of MUC5B^[4].However,there is an association between the MUC5B gene,which encodes mucins in the airway,and IPF,contradicting the fact that the etiology of IPF is generally thought to be limited to peripheral alveolar origin.However,MUC5B deposition was found in the distal bronchus and honeycomb cysts of IPF^[5],but its role and mechanism in IPF remain to be studied.Notably,the penetrance of SNP in the MUC5B gene promoter region is low,which increases the possibility that other factors are responsible for MUC5B post-transcriptional regulation.Micro RNA（mi RNA/mi R）is small endogenous non-coding RNA that can induce specific degradation or translation inhibition by binding to the 3’-untranslated region of target gene m RNA,thereby regulating gene expression and playing a key role in various biological processes,including the regulation of EMT processes.Because multiple mi RNAs can also target the same transcript,mi RNA regulatory networks are complex.To date,a small number of mi RNA targets have been validated,and the exact function of most mi RNAs remains to be elucidated.Mi R-221 is one of the mi RNAs that have attracted more attention in recent years,which is involved in the regulation of EMT in various tumors and is associated with organ fibrosis.However,whether mi R-221 can regulate EMT in pulmonary fibrosis and its mechanism are unclear.Therefore,the mechanism of MUC5B in the pathogenesis of IPF and the exploration of post-transcriptional regulatory factors will be crucial to prevent the occurrence of pulmonary fibrosis.Contents:Part I Expression level of MUC5B in pulmonary fibrosis and its correlation with EMTExperimental method:1.Public database（GEO）was mined according to the scheduling standard,suitable data sets were selected,and the MUC5B level of the target molecule was analyzed to evaluate the expression level of MUC5B in lung tissue and serum of patients with pulmonary fibrosis.2.Serum samples of patients with pulmonary fibrosis and normal controls were collected,and the level of MUC5B was detected by ELISA.The expression level of MUC5B in the dataset was verified.3.One-time intratracheal infusion of bleomycin（BLM）induced pulmonary fibrosis in mice.The living state and body weight of mice were recorded.HE,Masson,immunofluorescence double labeling and immunohistochemical staining were performed on the retained lung tissue.The expression levels of MUC5B,α-SMA,Vimentin and E-cadherin were detected by RT-q PCR and Western blot.To evaluate the expression of MUC5B in mice with pulmonary fibrosis and its effect on EMT.4.TGF-β1 induced human alveolar epithelial cells（A549）to construct EMT model.The levels of MUC5B andα-SMA,Vimentin,E-cadherin were detected.To evaluate the effect of EMT on MUC5B expression in alveolar epithelial cells.Experimental results:1.By analyzing the data set,it was found that there were significant differences in gene expression profiles between patients with pulmonary fibrosis and healthy controls.However,the expression of MUC5B was increased in lung tissue and serum of patients with pulmonary fibrosis.2.Clinical sample analysis:Through the collection and analysis of clinical samples,it was confirmed that MUC5B was elevated in the serum of patients with pulmonary fibrosis,which verified the results of bioinformatics analysis.3.In animal experiments,after BLM administration,mice showed shortness of breath,decreased appetite,decreased activity,and weight loss.Combined with the pathological manifestations of lung tissue,the successful construction of mouse pulmonary fibrosis model could be judged.RT-q PCR and Western blot results showed that the expression of MUC5B was increased,Vimentin andα-SMA were increased,and the expression of E-cadherin was decreased.Immunohistochemical staining of lung tissue showed that MUC5B was expressed in cytoplasm and increased in mice with pulmonary fibrosis.Double immunofluorescence labeling of lung tissue showed the expression of MUC5B in pro-Spc labeled type II alveolar epithelial cells.4.In the cell experiment,the morphology of A549 cells changed from polygonal to long spindle after induction of EMT.RT-q PCR and Western blot results showed that the expression of MUC5B was increased,Vimentin andα-SMA were increased,and E-cadherin expression was decreased,and the changes were time-and concentration-dependent.PartⅡRole of MUC5B in pulmonary fibrosis Experimental method:1.Mice were given endotracheal instillation of AAV-MUC5B virus.21 days later,BLM was injected into the trachea.The living state and body weight of mice were recorded during the period.The entry of virus into lung tissue was observed by imaging of small animals and frozen sections of lung tissue.The content of hydroxyproline in lung tissue was detected.HE and Masson staining were performed,and the levels of Collagen-Ⅰ,α-SMA,Vimentin,E-cadherin,Fibronectin and EGFP were detected by immunohistochemical staining,immunofluorescence double-label staining,Western blot and RT-q PCR,and the expression of mucin was evaluated by PAS staining.PAS staining was used to evaluate mucin expression and its effect on pulmonary fibrosis.2.A lentiviral vector overexpressing MUC5B gene was constructed using CRISPR/Cas9.And infected A549 cells.Fluorescence microscopy observed the infection efficiency.The overexpression level of MUC5B was detected by RT-q PCR and Western blot.To evaluate whether stable cell lines overexpressing MUC5B were successfully constructed.Successful overexpression of MUC5B induces EMT.The levels ofα-SMA,Vimentin and E-cadherin were detected by RT-q PCR and Western blot,assess the impact on EMT.3.Transfected A549 cells with si RNA silenced MUC5B expression.The transfection efficiency was observed by fluorescence microscopy,and the inhibition level of MUC5B was detected by RT-q PCR and Western blot.After successfully silencing MUC5B,EMT was induced,and the expression levels ofα-SMA,Vimentin and E-cadherin were detected by RT-q PCR and Western blot.Assess the impact on EMT.Experimental results:1.After endotracheal infusion of AAV in mice,fluorescence signals can be observed in lung tissue through live imaging of small animals.Enhanced green fluorescence was also observed in frozen sections of lung tissue.The results indicated that AAV successfully entered mouse lung tissue.The results of RT-q PCR and Western blot indicated that the expression of MUC5B was decreased,and the model of low expression of MUC5B in mouse lung tissue was successfully constructed.2.After BLM intervention,the symptoms of mice with low expression of MUC5B were reduced,and the weight loss was not obvious.Mild hyperemia and edema of lung tissue were observed after dissection.Hydroxyproline assay showed the decrease of collagen deposition.The pulmonary histopathological findings suggested that alveolitis was alleviated and the degree of fibrosis was alleviated.The results of immunohistochemical staining,RT-q PCR and Western blot indicated that the levels of Collagen-Ⅰ,α-SMA,Vimentin and Fibronectin were decreased,while the levels of E-cadherin were increased.PAS staining showed decreased expression of mucin.3.Fluorescence microscopy observed lentivirus successfully infected A549 cells.The results of RT-q PCR and Western blot indicated that the expression of MUC5B was increased.The results indicated that the stable cell line overexpressing MUC5B was successfully constructed.After administering TGF-β1 to induce EMT,Western blot and RT-q PCR results showed thatα-SMA and Vimentin expression increased significantly.The level of E-cadherin was significantly reduced,which aggravated EMT.4.After successful transfection of si RNA into A549 cells by fluorescence microscopy,RT-q PCR and Western blot showed that the expression of MUC5B was silenced.After administering TGF-β1 to induce EMT,Western blot and RT-q PCR results showed that the expression ofα-SMA and Vimentin decreased,while the levelXof E-cadherin increased.Reduced EMT degree.PartⅢMi R-221-5p targets the role and mechanism of MUC5B in the formation of pulmonary fibrosisExperimental method:1.Using mi RNA bioinformatics target prediction tools,to predict the binding sites of mi R-221-5p and MUC5B,the targeting relationship between the two was verified by double luciferase reporter gene assay.2.The level of mi R-221-5p after EMT induction was detected by FISH and RT-q PCR.A549 cell models with high or low expression of mi R-221-5p mimic and mi R-221-5p inhibitor were constructed and EMT was induced.Levels ofα-SMA,Vimentin,E-cadherin,Collagen-Ⅰ,Fibronectin were detected by ELISA,RT-q PCR,Western blot and immunofluorescence staining.To determine the effect of mi R-221-5p on EMT of alveolar epithelial cells.The level of MUC5B was detected by Western blot analysis to determine the effect of mi R-221-5p on MUC5B expression.3.A549 cells overexpressing MUC5B co-transfected mi R-221-5p mimic.The levels ofα-SMA,Vimentin and E-cadherin were detected by Western blot.To evaluate the effect of mi R-221-5p on EMT of alveolar epithelial cells through MUC5B.4.Western blot tests for p-Smad3 levels,to determine the influence of TGF-β1/Smad3 signaling pathway.Experimental results:1.There are binding sites between MUC5B and mi R-221-5p.Dual luciferase gene reporting experiments confirmed the targeting relationship between the two.2.With the prolonged stimulation time and increased concentration of TGF-β1,the expression of mi R-221-5p in A549 cells decreased gradually.Overexpression of mi R-221-5p alleviated TGF-β1-induced EMT and inhibited the expression of MUC5B in A549 cells.Silencing the expression of mi R-221-5p could promote EMT and up-regulate the expression of MUC5B.Overexpression of MUC5B can antagonize the inhibition of EMT induced by overexpression of mi R-221-5p.3.Overexpression of mi R-221-5p can decrease the expression of p-Smad3.Silence of MUC5B also decreased the expression of p-Smad3.At the same time,mi R-221-5p and MUC5B were overexpressed,and p-Smad3 expression was increased.The TGF-β1/Smad3 signaling pathway was activated.Research conclusion:1.The expression of MUC5B was increased in patients with pulmonary fibrosis and in mice with bleomycin induced pulmonary fibrosis.The occurrence of pulmonary fibrosis is related to the ectopic expression of MUC5B in type II alveolar epithelial cells.2.Silencing MUC5B can inhibit collagen deposition in lung tissue of mice with pulmonary fibrosis.Inhibit epithelial-mesenchymal transformation and improve pulmonary fibrosis.Overexpression of MUC5B can promote EMT in alveolar epithelial cells.Low expression of MUC5B can reverse this effect.3.Mi R-221-5p alleviates EMT in alveolar epithelial cells by targeting inhibition of MUC5B.Overexpression of MUC5B may promote EMT in alveolar epithelial cells through activation of TGF-β1/Smad3 signaling pathway,play a role in promoting fibrosis.