| Pulmonary fibrosis(PF)is a very common chronic,incurable disease,which is characterized by lung tissue scarring and the destruction of lung tissue structure.Despite the development of various detection methods and clinical treatments,PF remains a major public health problem that inevitably leads to a decline in lung function,progressive respiratory failure,and high mortality.Increasing evidence suggests that a possible mechanism for the pathogenesis of PF involves epithelial–mesenchymal transition(EMT),a transdifferentiation process in which epithelial cells are ultimately converted into motility mesenchymal cells with increased collagen deposition and other extracellular matrix components characteristic.Tracheobronchial tuberculosis(TBTB)is defined as the tubercle bacilli infection of the trachea and/or bronchus and is one of the most common complications of TBTB.the current treatment methods for tracheobronchial stenosis caused by TBTB are very limited.Epithelial-mesenchymal transition,characterized with a mesenchymal phenotype and loss of epithelial characteristics is a major contributor to the pathogenesis of fibrosis.Micro RNAs(mi RNAs/mi Rs)are small endogenous non-coding RNAs that can regulate gene expression by binding to the 3'-untranslated region(3'-UTR)of target m RNA to block translation and/or accelerate the degradation of target m RNA.There is increasing evidence that mi RNAs play critical roles in various biological processes,including cell proliferation,differentiation,apoptosis and regulation of the EMT process.The mi RNA miR-320a-3p is known to be involved in the regulation of EMT,and,recently,numerous studies have shown that it plays important roles in the regulation of the EMT process in various tumors.However,whether miR-320a-3p can regulate EMT in PF and TBTB,and whether there is a common mechanism in this process is unclear.In this study,we investigated the role of miR-320a-3p in EMT of pulmonary fibrosis and TBTB-induced airway fibrosis(stenosis),and their underlying mechanism were elucidated through cell model experiments.Objective1.Through public database mining,the expression levels of miR-320a-3p in the datasets were analyzed.Next,the expression levels of miR-320a-3p were measured between pulmonary fibrosis samples and normal control samples,and also between TBTB samples and normal control samples.2.To clarify the role of miR-320a-3p played in EMT,miR-320a-3p mimic was used to interfere with the EMT models of A549 and BEAS-2B cells induced by TGF-?1.3.The mechanisms of miR-320a-3p on EMT were preliminarily elucidated at the cellular level,and the clues about new therapeutic targets for FP and airway fibrosis(stenosis)caused by TBTB were provided.Methods1.According to the criteria,the public databases Gene Expression Omnibus(GEO)and Array Express were mined,and the expression levels of miR-320a-3p in these datasets were analyzed.2.Clinical samples of PF,TBTB and normal control tissue were collected.The expression levels of target mi RNA were measured by q PCR assay.3.Construct the cell models,and the expression levels of miR-320a-3p in cell models were determined by q PCR assay.Transfect the cell model with miR-320a-3p mimic and then stimulate with TGF-?1,and the effect of miR-320a-3p on EMT was determined by q PCR,Western blot and immunofluorescence staining assays.4.The direct target genes of miR-320a-3p were predicted by bioinformatics tools and confirmed by Dual-luciferase reporter assay.The related mechanism of miR-320a-3p on EMT was elucidated by Western blot assay.Results1.Screening of datasets from public databases,and the expression levels of mi RNA in clinical samples and cell models were analyzed.According to the inclusion criteria,GEO and Array Express databases were retrieved,and a total of 3 datasets(174 samples)were included in.Statistical analysis showed that,in these data sets,the expression levels of miR-320a-3p were different between pulmonary fibrosis samples and normal control samples.Next,the pulmonary fibrosis samples and normal control samples were collected.By detecting the expression levels of miR-320a-3p in the clinical samples,we found that compared with the normal control samples,the expression levels of miR-320a-3p in pulmonary fibrosis were significantly reduced.Subsequently,the expression levels of miR-320a-3p in TBTB samples and normal control samples were measured too,and the results showed that the expression levels of miR-320a-3p in TBTB samples were significantly lower than that in normal control samples.Therefore,we inferred that the expression levels of miR-320a-3p were correlated with the disease in the fibrosis of lung and airway,and it was hypothesized that the increasing expression levels of miR-320a-3p might affect pulmonary and airway fibrosis.Next,A549 and BEAS-2B cells were stimulated with TGF-?1 to construct EMT cell models of PF and TBTB.TGF-?1 was used to stimulate A549 and BEAS-2B cells at different concentrations(0,1,3,5,and 10ng/ml)for 48 h and it was found that compared with the control group the expression of miR-320a-3p in TGF-?1-stimulation A549 cells and BEAS-2B cells were significantly decreased in a concentration-dependent manner.After stimulating A549 cells and BEAS-2B cells with 10ng/ml TGF-?1 at different time gradients(0,24,48 h),the expression level of miR-320a-3p was measured.It was found that compared with the control group,the expression levels of miR-320a-3p decreased most obviously at 48 h.2.Mi R-320a-3p inhibited TGF-?1-induced EMT and cell migration ability.We treated A549 cells and BEAS-2B cells with miR-320a-3p mimic,and then stimulated cells with TGF-?1.The results of q PCR,western blot and immunofluorescence assays showed that TGF-?1 stimulation decreased the expression of epithelial marker E-cadherin,while increased the expression of mesenchymal marker Vimentin and ?-SMA in both A549 cells and BEAS-2B cells.Compared with TGF-?1-treated group,miR-320a-3p mimics group significantly increased the expression of E-cadherin and decreased the expression of vimentin and ?-SMA.For BEAS-2B cells,we further carried out real-time cell analyzer(RTCA)migration ability assay and scratch assay.The results show that compared with the control cells,the BEAS-2B cells pretreated with TGF??1 showed a higher motility.Transfection of miR-320a-3p mimic significantly reduced the migration ability of BEAS-2B cells and decreased the migration ability of TGF-?1 pretreated cells close to the control level.3.The mechanism of miR-320a-3p inhibiting EMT in lung and airway epithelial cellsBy using bioinformatics tools to predict the target genes of miR-320a-3p,we found that STAT3 was a potential target gene of miR-320a-3p.Through dual-luciferase assay,we confirmed that miR-320a-3p could bind to the 3 '-UTR region of wild-type STAT3,but could not bind to the 3'-UTR region of mutated STAT3.Next,we measured the expression levels of STAT3 in miR-320a-3p mimictransfected A549 cells and BEAS-2B cells,and it was found that compared with TGF-?1 treatment group miR-320a-3p mimic treatment significantly reduced the expression of STAT3.These results suggested that miR-320a-3p may inhibit EMT in lung and airway epithelial cells by targeting STAT3.To further confirm that miR-320a-3p inhibited TGF-?1-induced EMT in A549 cells and BEAS-2B cells through a STAT3-dependent mechanism.First,A549 cells and BEAS-2B cells were transfected with STAT3 si RNA and NC si RNA and then treated them with TGF-?1(10ng/ml)for 48 h.The results of Western blot assay showed that,compared with the control group,STAT3 si RNA reduced the expression of vimentin and ?-SMA,while the expression of E-cadherin was significantly increased.Then we performed a rescue experiment,miR-320a-3p mimics and HBLV-h-STAT3 or HBLV-NC were co-transfected into A549 cells and BEAS-2B cells.The results showed that Compared with mock-transfected cells,HBLV-h-STAT3 significantly increased the expression of STAT3.In addition,the upregulation of STAT3 inhibited the effect of the miR-320a-3p mimic on the expression of E-cadherin and significantly increased the expression of vimentin,?-SMA.For this study further,miR-320a-3p mimic,si-STAT3,and miR-320a-3p mimic combined with HBLV-h-STAT3 were transfected into cells respectively.The results showed that miR-320a-3p mimic and si-STAT3 could significantly reduce the expression levels of p-Smad3 in cells,and the co-transfection of miR-320a-3p mimic with HBLV-h-STAT3 reversed the reduction of p-Smad3 expression caused by miR-320a-3p mimic.ConclusionBased on public database mining and clinical sample testing,we found that the expression levels of miR-320a-3p were significantly reduced in the pulmonary and airway fibrotic tissues.The expression levels of miR-320a-3p are related to the occurrence and development of pulmonary and airway fibrosis.Mi R-320a-3p can inhibit the epithelial-mesenchymal transformation of A549 cells and BEAS-2B induced by TGF-?1 stimulation.The mechanism may be related to the STAT3/ Smad3 signaling pathway.Although the disease categories of PF and TBTB are different,miR-320a-3p has a common mechanism in inhibiting EMT of both lung and airway fibrosis.This study provides a theoretical basis for miR-320a-3p as a possible therapeutic target for pulmonary and airway fibrosis. |
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