Effects Of FOXO3a On TGF-?-induced Epithelial-mesenchymal Transition Of Human Bronchial Epithelial Cells

1 BackgroundInterstitial lung disease(ILD)is a series of lung diffuse lesions which could be involved in pulmonary interstitial,airway and alveolar.It is always characterized by various types of extracellular matrix deposition and cell infiltration at the end o f the bronchioles,The alveolar-capillary function unit is gradually damaged and leads to diffuse pulmonary fibrosis and cellular lung.The ILD is poorly treated and has high mortality.Especially,idiopathic pulmonary fibrosis(IPF),is the most harmful one.its pathophysiological process have not been fully elucidated.There is still no specific and effective treatment to prevent or reverse the progress of its course.Currently,the pathogenesis of IPF is recognized as the damage of alveolar and bronchial epithelial cells [1-3].Epithelial-mesenchymal transition(EMT)plays an important role in the pathogenesis of ILD and it is considered to be an important feature of pulmonary fibrosis.Airway epithelial cells are differentiated into mesenchymal cells.The adhesion structure of epithelial cells is instead by Type ? collagen and fibronectin synthesis.The above factors become the basis of changes in pulmonary fibrosis.The mechanism of EMT in pulmonary fibrosis is still unknown.Some studies have confirmed it is connected with EGFR/Akt,TGF-beta/Smad related signal transduction pathways[4-5].To identify the key link can effectively curb the progress of ILD.Transforming growth factor(TGF-?)is an important molecule that regulates cell proliferation,differentiation and other functions,and it is also an important initiating factor in the EMT of pulmonary fibrosis.Studies have shown that TGF-? can induce EMT through Smad pathway in tissue cells,leading to the production of tissue and organ fibrosis[6-7].It has been confirmed both in vivo and in vitro experiments.The forkhead box protein(FOX)family is a transcription factor with a wing-like spiral structure in a DNA-linked region,which is associated with body development,cellular immunity,substance metabolism and cancer.Recent studies have shown that the FOX family is an important transcription factor that regulates epithelial-mesenchymal transition[ 8 ],Its important member,forkheadbox protein O3a(Foxo3a),is involved in the development of fibrosis.The study shows that FOXO3 a plays an important role in TGF-?-induced renal epithelial transdifferentiation [9].Another study shows that FOXO3 a is also involved in the pulmonary fibrosis EMT process,which may be related to the reduction of the the transdifferentiation gene,Snail.O ur previous study showed that the use of gefitinib could down-regulation of TGF-? pathway,increased FOXO3 a expression,and significantly reduced bleomycin-induced pulmonary fibrosis cell proliferation.This study suggests that FOXO3 a is a key node in the process of EMT,which is involved in the EMT process of TGF-? induced pulmonary fibrosis.Therefore,for the further study of the mechanism of transcriptional regulation of FOXO3 a in TGF-? induced EMTchanges,this study will investigate the role of FOXO3 a in the epithelial mesenchymal transition process of pulmonary fibrosis,and to lay the experimental foundation for further studise.2 Objective 2.1 Use TGF-? to induce human bronchial epithelial cells,evaluate the expression of epithelial cell phenotype and stromal cell phenotypic markers,and establish EMT model of ILD in vitro.2.2.Investigate the expression of FOXO3 a in the process of EMT.2.3 Use gene recombination technique to investigate the role of enhanced FOXO3 a expression in EMT of pulmonary interstitial fibrosis.3 Methods and materials 16 HBE cells derived from bronchial epithelial cells were supplied by the laborato ry of General Hospita of Guangzhou Military Region.16 HBE cells were cultured in a high glucose medium with 10% inactivated calf serum in RPMI-1640 medium.3.1 Construction of epithelial-mesenchymal transition model of bronchial epithelial cells 3.1.1 Expression of ?-SMA and E-cadherin mRN A and protein in bronchial epithelial cells induced by TGF-? 16 HBE cells were cultured in vitro with 10% FBS medium.The third generation cells were inoculated on the 6 hole plate and cultured for 24 h after 10%FBS,and then cultured in 1%FBS for 24 h.The 16 HBE cells were divided into two groups:(1)control group,10 ? g / L TGF-? group,the total RNA and total protein were extracted by 24 h,48h and 72 h respectively,and the expression of E-cadherin and-SMA were detected by PC R and Western Blot.3.1.2 Use immunohistochemistry to detect the localization and expression of E-cadherin and ?-SMA on 16 HBE cells 16 HBE cells were cultured in vitro with 10% FBS medium.The cells were inoculated on 6-well plates and cultured in 10% FBS for 24 h.After 1% FBS culture for 24 h,the cells were divided into two groups:(1)control group,10 ? g/L TGF-? group,Immunohistochemical technique was used to detect the localization and expression of E-cadherin and ?-SMA.3.2 Use PCR and Western Blot to detect the expression of FOXO3 a mRNA and Protein in 16 HBE Cells during EMT 16 HBE cells were cultured in vitro with 10% FBS medium.The third generation cells were inoculated on the 6 hole plate and cultured for 24 h after 10%FBS,and then cultured in 1%FBS for 24 h.The 16 HBE cells were divided into two groups:(1)control group,10 ? g / L TGF-? group,the total RNA and total protein were extracted by 24 h,48h and 72 h respectively,and the expression of FOXO3 a were detected by PCR and Western Blot..3.3 Effect of enhanced FOXO3 a expression on cell during EMT.3.3.1 Construction and verification of FOXO3 a plasmid.The sequence of human FOXO3 a gene was obtained from Gen Bank,primers were designed and FOXO3 a gene fragment was amplified by PCR.The linear vector was obtained by restriction enzyme digestion,and the reaction was carried out by using the linear carrier and FOXO3 a gene amplification product,and then the recombinant vector was used to realize the in vitro cyclization of the linear vector and FOXO3 a gene fragment.The recombinant product can be directly transformed,identified by PC R and The positive clones were sequenced and analyzed.The correct clone liquid was expanded and extracted to obtain high purity plasmid.3.3.2 Detect the expression of ?-SMA,E-cadherin mRNA and protein of enhanced FOXO3 a expression cells during EMT.The cells were divided into six groups: normal control group,FOXO3 a group,negative plasmid group,TGF-? induction group,TGF-? + FOXO3 a group and TGF-? + negative plasmid group.Transfection was performed according to the kit instructions.After 48 h,the total RN A and total protein were obtained.Use PCR and Westernblot to detect the expression of-SMA,E-cadherin mRNA and protein in each group.3.4 Statistical methodStatistical analysis was performed using spss20.0 software.Metrological data were expressed as mean ± standard deviation(X±S).The data of each group were tested by homogeneity of variance.One-way ANOVA was used to compare the meanings of the groups.Variance,multiple comparison using LSD test,if the variance was not the same,multiple comparison using Dunnett's T3 test,P <0.05 difference was statistically significant.4 Result 4.1 Construction of epithelial-mesenchymal transition model of bronchial epithelial cells 4.1.1 Expression of ?-SMA and E-cadherin mRN A and protein in bronchial epithelial cells induced by TGF-?The expression of ?-SMA mRNA in TGF-? group was significantly higher than that in control group(P <0.05).There was no significant difference in negative control group(P> 0.05)(P <0.05).There was no significant difference between the negative control group and the negative control group(P> 0.05).The difference was statistically significant(P <0.05).The expression of ?-SMA protein in TGF-? group was significantly higher than that in control group(P <0.05).There was no significant difference in negative control group(P> 0.05)(P <0.05).There was no significant difference between the negative control group and the control group(P> 0.05).The difference was statistically significant(P <0.05).4.1.2 Use immunohistochemistry to detect the localization and expression of E-cadherin and ?-SMA on 16 HBE cellsT The results of immunohistochemistry showed that both E-cadherin and-SMA were distributed in the cytoplasm,E-cadherin was highly expressed in normal 16 HBE cells,and the expression of E-cadherin was significantly decreased after 48 h of TGF-?(10?g / L)stimulation.The expression of ?-SMA in normal 16 HBE cells was significantly increased after 48 h of TGF-? stimulation.4.2 Use PCR and Western Blot to detect the expression of FOXO3 a mRNA and Protein in 16 HBE Cells during EMTThe expression of FOXO3 a mRNA in 10?g / L TGF-? group decreased with time,the difference was statistically significant(P <0.05);the negative control group was not statistically significant(P> 0.05)The expression of FOXO3 a protein in 10?g / L TGF-? group decreased with time,the difference was statistically significant(P <0.05);the negative control group was not statistically significant(P> 0.05)4.3 Effect of enhanced FOXO3 a expression on cell during EMT.4.3.1 Construction and verification of FOXO3 a plasmid.4.3.1.1 Amplification and identification of full-length FOXO3 a CDS by PCR The sequence of the target gene was identical with that of human FOXO3 a Pubmed gene in the CDS gene sequence.4.3.1.2 Single colony PCR verification Take single colony to PCR electrophoresis,the electrophoresis results are correct.4.3.1.3 Plasmid sequencing results The GV230-FOXO3 a plasmid was sequenced by the NCBI plasmid,and the results were correct.4.3.1.4 Plasmid Transfection Fluorescence Verification The recombinant plasmid was transfected into 16 HBE cells after 12 h,normal control group showed no green fluorescence,GV230-FOXO3 a plasmid group and negative plasmid transfected group can be observed green fluorescence,proved the transfected and expressed the protein in the cell..4.3.2 Detect the expression of ?-SMA,E-cadherin mRNA and protein of enhanced FOXO3 a expression cells during EMT.The expression of ?-SMA mRNA and protein in TGF-? + FOXO3 a plasmid group was significantly lower than that in TGF-? + negative group and TGF-? group,the difference was statistically significant(P <0.05)(P> 0.05).There was no significant difference between the normal control group and the FOXO3 a group and the negative plasmid group(P> 0.05).The expression of E-cadherin mRNA and protein in TGF-? + FOXO3 a plasmid group was significantly higher than that in TGF-? + negative group and TGF-? group,the difference was statistically significant(P <0.05)(P> 0.05).There was no significant difference between normal control group,FOXO3 a group and negative plasmid group(P> 0.05).There was no significant difference between the control group and the control group(P> 0.05).5 Conclusion TGF-?-induced bronchial epithelial cells can successfully simulate the process of EMT in vitro.FOXO3 a inhibits TGF-?-induced epithelial-mesenchymal transition in bronchial epithelial cells...