Six1 Regulates The Interstitial Transformation And Fibrosis Of Human Bronchial Epithelial Cells 16HBE Through The TGFβ1/Smad Signaling Pathway

Part Ⅰ The effect of overexpression Six1 in human bronchial epithelial cells on epithelial-mesenchymal transition and fibrosisObjective:To investigate the effect of over expression Six1 on the epithelial-mesenchymal transition（EMT）and fibrosis of 16 HBE cells.Methods: Six1（NM₀05982）-GV146 over expression plasmid glycerol were cultured in LB medium.The plasmid was extracted and its concentration was determined after endotoxin was removed.16 HBE cells were cultivated in vitro and transfected with Six1 by using LipofectamineTM2000,and set up blank control group（using LipofectamineTM2000 only）and negative control group（using negative gene with LipofectamineTM2000）.After cells were transfected for 48 hours,observed the morphological changes of cells by using inverted microscope;The expression of Six1,E-cadherin,vimentin,α-SMA,keratin m RNA were detected by Real-Time PCR;And the expression of Six1,E-cadherin,vimentin,α-SMA,keratin protein were detected by Western blot;RT-PCR and Western blot were used to detect the expression of fibronectin m RNA and protein in each group.Results:（1）Observed the cells using inverted microscope: positive group show cell gap increased with fusiform shape compared with the negative control group and blank control group;（2）Compared with negative control group and blank control group,the expression of Six1 in positive group were increased through RT-PCR and Western blot,the differences were significant（F=881.422、78.376,P<0.01）.While there were no significant between negative control group and blank control group（t=0.002、0.051,P>0.05）;（3）The expression of E-cadherin and keratin were decreased significantly in m RNA（F=20.370、15.232,P <0.01）and protein（F=17.123、10.713,P<0.01、P<0.05）,while α-SMA,vimentin were increased significantly in m RNA（F=9.791、36.085,P <0.01）and protein（F=44.161、11.397,P<0.01）,these differences were statistically significant.There were no significant differences in the expression of m RNA（t=0.0.056、0.461、0.0.094、0.277,P>0.05）and protein（t=0.894、0.730、0.080、0.185,P>0.05）between the negative control group and the blank control group;（4）The expression of fibronectin was also increased in m RNA and protein（F=230.003、23.256,P <0.01）,these differences were statistically significant.There were no significant difference between negative control group and blank control group（t=0.376、1.151,P>0.05）.Conclusion: Over expression Six1 can change the phenotype of 16 HBE cells,which also increased the intercellular space.After detection,the character products of epithelial cells were reduced,while the character products of mesenchymal cells were increased,as well as the expression of fibronectin increased.Six1 may had an effect on the EMT and fibroblasts changes of 16 HBE cells.Part Ⅱ Six1 promotes epithelial-mesenchymal transition 16 HBE cells by TGFβ1/Smad signal pathwayObjective:To further explore the mechanism of Six1 on human bronchial epithelial-mesenchymal transition and fibrosis changes by regulating TGFβ1/Smad signal pathway,and to explore the role of TGFβ1/Smad signal pathway in it.Methods:（1）The glycerol bacteria containing Six1（NM₀05982）-GV146 plasmid were expanded and extracted.16 HBE cell were cultured in vitro.Great growth cells were divided into Six1 group（adding Six1-GV146 plasmid）,negative control group（adding synthetic nonsense-GV146 plasmid）and blank control group（only adding transfected liposome）.48 hours later,TGFβ1 and phospho–Smad2 protein were determined by Western blot.（2）16HBE cells transfected with Six1（NM₀05982）-GV146 plasmid were divided into inhibitor group and Six1 control group.6 hours after transfection,inhibitor group was treated with 10% FBS medium containing TGFβ1 pathway inhibitor（10nmol/L）,while the Six1 control group only replaced the 1640 medium with 10% FBS.24 hours after the addition of inhibitor,morphological changes were observed by inverted microscope;The expressions of E-cadherin,α-Smooth Muscle Actin,TGFβ1,phospho–Smad2 protein and fibronectin in each group were measured by Western blot.Results:（1）After over expressed Six1 in 16 HBE cells,the expression of TGFβ1 and phospho–Smad2 protein in Six1 group were significantly higher than that in the negative control group and blank group（F=63.323、39.948,P<0.05）.There were no significant differences in the expression of TGF β 1 and phospho-Smad2 between the negative control group and the blank control group（t=0.859、0.445,P>0.05）.（2）Phenotypic changes observed by inverted microscope after inhibited for 24h: The cells gap of the inhibitor group was significantly smaller than that of the Six1 control group,and the cells morphology was similar to pebble-like monolayer cells,while the cells of Six1 control group showed fusiform changes,the intercellular connection were not tight,and the cells gap were large.（3）Compared with the Six1 control group,the expression of TGFβ1 and phospho–Smad2 protein in inhibitor group decreased significantly（t=4.454、t=7.104,P<0.05）.E-cadherin in group inhibitor increased（t=5.367,P<0.05）,and the expression of α-Smooth Muscle Actin decreased significantly（t=7.126,P<0.05）compared with the Six1 control group.These differences were statistically significant.（4）The expression of fibronectin in inhibitor group is significant lower than that in Six1 control group（t=9.422,P<0.05）,this difference was statistically significant.Conclusion:（1）Up-regulated the expression of Six1 in 16 HBE cells increased the expression of TGFβ1/ Smad signal pathway.（2）Further inhibited of TGFβ1/ Smad signal pathway,the changes of EMT and fibrosis were significantly inhibited.Six1 induced EMT and firbrosis changes in 16 HBE cells,which may be caused by TGFβ1/ Smad signaling pathway.