Effects of 5-azacytidine on radiation-induced epithelial-mesenchymal transition in rat alveolar type II epithelial cell line

Objective To investigate the effects of 5-azacytidine on radiation-induced epithelial-mesenchymal 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 radiation-induced 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, GSK3β, and p-GSK3β (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 E-cadherin 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-GSK3p in RLE-6TN cells. Key words: 5-Azacytidine; X-ray irradiation; RLE-6TN cell line; Epithelial-mesenchymal transition; Rat