Transcriptional Factor Snail Mediate Epithelial-mesenchymal Transition In Human Bronchial Epithelial Cells Induced By Silica

Objective:To explore whether transcriptional factor Snail mediate Epithelial-mesenchymal transition(EMT) in Human bronchial epithelial cells(HBE) induced by silica.Methods:Using HBE induced by 200μg/ml SiO2 as a model:(1)At different time points (0,1,6,12,18,24h), the morphological changes of HBE were observed by inverted microscope.(2) At different time points (0,24h), the ultrastructure changes of HBE were observed by electron microscopy.(3)At different time points (0,1,6,12,18,24h), Western blotting was used for detecting the total protein expression of Snail.(4) At different time points (0,1,6,12,18,24h), Western blotting was used for detecting the nucleoprotein expression of Snail.(5)At different time points (0,1,6,12,18,24h), Immunofluorescence was used for detecting the localization of Snail in the nuclear.(6) At different time points (0,12,18h), Electrophoretic Mobility Shift Assay(EMSA) was used for detecting the Snail DNA binding activity.(7) After siRNA interfered, Western blotting was used for detecting Snail, Vimentin, smooth muscle actin (a-SMA) and E-cadherin (E-cad) expression.Reslut:(1)Inverted microscope shows that HBE cells were changed from cobblestone into a spindle-shaped.(2) Electron microscopy shows that control cells were round, and the surfaces were abundanted in microvilli; after incubation of HBE cells with 200μg/ml SiO2,cells became elongated and the number of microvilli decereased,while the number of microtubule-like structures increased in cytoplasm. (3)Using Western blotting, the expression of the total protein of Snail increased in HBE after 1 hours,it reached peak at 18 hours,the protein levels of Snail were significantly different between 12 hours/18 hours group and control group(2.88±0.63/4.51±1.15 vs 0.57±0.04,P＜0.05).(4)Using Western blotting,the expression of the nucleoprotein of Snail increased in HBE after 1 hours,it reached peak at 12 hours, the protein levels of Snail were significantly different between 12 hours/18 hours group and control group (4.51±0.81/2.76±0.77 vs 0.67±0.15,P<0.05). (5)Immunofluorescence shows that the weak fluorescence of cytoplasm in control group and strong expression of fluorescent in nucleus of experimental group.(6)EMSA shows:Snail DNA binding activity expression levels of 12 hours group(0.020±0.00047) and 18 hours group (0.023±0.0011) were significantly higer than control group (0.0014±0.00026) (P<0.05).(7)siRNA interfered shows:①the protein levels of Snail after RNA interference were significantly different between SiO2 group and siRNA group(20.02±1.44 vs 6.73±1.03,P<0.05),the inhibition ratio was 66.38%.②the protein levels of E-cad after RNA interference were significantly different between SiO2 group and siRNA group(0.18±0.03 vs 0.84±0.01,P＜0.05).③the protein levels of Vimentin and a-SMA after RNA interference were significantly different between SiO2 group and siRNA group(13.12±1.09 and 2.35±0.45 vs 2.33±0.54 and 0.23±0.05,P<0.05),the inhibition ratio were 82.24% and 90.21%.Conclusion:(l)SiO2 could induce the epithelial-mesenchymal transition(EMT) of HBE cells;(2) HBE cells induced by SiO2 could enhance the activation of Snail;(3)Snail could mediate EMT in HBE cells induced by SiO2.