ABSTRACT
Objective To investigate the effects of 5-azacytidine on radiation-induced epithelialmesenchymal transition in rat alveolar type Ⅱ epithelial cell line (RLE-6TN) and explore their working mechanisms,and to provide experimental evidence for the potential drug-based treatment of radiationinduced pulmonary fibrosis.Methods RLE-6TN cells were cultured in vitro and divided into four groups according to the experimental purposes:control group (C),radiation group (R),5-azacytidine group (A),and radiation followed by 5-azacytidine group (R + A).The microstructural changes in cells were determined by transmission electron microscopy.Inverted phase-contrast microscopy revealed the morphological changes in cells.The mRNA expression levels of E-cadherin and α-SMA were measured by quantitative real-time polymerase chain reaction (qRT-PCR).The protein expression levels of E-cadherin,GSK3 β,and p-GSK3 β (Ser9) were measured by Western blot.The one-way analysis of variance was used for pairwise comparison.Results The cells in group R became spindle-like.Similar morphological changes were not observed in cells in group R + A.Osmiophilic lamellar bodies disappeared at last in cells in group R.RT-PCR results showed that compared with group C,group R had a significantly lower mRNA expression level of E-cadherin ((0.23 ± 0.06) vs.(1.00 ± 0.00),P =0.002)) and a significantly higher mRNA expression level of α-SMA ((2.91 ± 0.01) vs.(1.00 ± 0.00),P =0.000)).However,compared with group R,group R + A had a significantly higher mRNA expression level of E-cadherin ((0.47 ± 0.05) vs.(1.00 ± 0.00),P =0.024)) but a significantly lower mRNA expression level of α-SMA ((2.50 ± 0.02) vs.(1.00 ±0.00),P =0.037)).The results of Western blot showed that the protein expression level of Ecadherin was significantly reduced ((0.07 ± 0.01) vs.(0.48 ± 0.02),P =0.028)),while the protein expression level of p-GSK3β was significantly increased in Group R than in Group C ((0.85 ± 0.04) vs.(0.23 ± 0.03),P =0.031)).However,compared with group R,group R + A had a significantly lower protein expression level of E-cadherin ((0.25 ± 0.00) vs.(0.07 ± 0.01),P =0.024)) and significantly less up-regulation of the protein expression level of p-GSK3β ((0.39 ± 0.03) vs.(0.85 ± 0.04),P =0.014)).Conclusions X-ray radiation can induce the epithelial-mesenchymal transition in epithelial cells.5-azacytidine suppresses radiation-induced epithelial-mesenchymal transition by inhibition of the activity of p-GSK3β in RLE-6TN cells.