Regulation of EMT in lung cancer cells by interaction of Hsp90 and SIRT1 / 国际肿瘤学杂志

ABSTRACT

Objective:To investigate the interaction between heat shock protein 90 (Hsp90) and silent mating-type information regulation 2 homolog 1 (SIRT1) and evaluate its effect on epithelial-mesenchymal transition (EMT) of lung cancer A549 cells.Methods:EMT model was established by treating lung cancer A549 cells with 5 μg/L transforming growth factor-β1 (TGF-β1), which was used as TGF-β1 group, and the normal lung cancer A549 cells were used as control group. The interaction between Hsp90 and SIRT1 in lung cancer A549 cells was detected by immunocoprecipitation method. The expression of Hsp90 gene was silenced by RNA interference technique, and the cells were divided into TGF-β1 group, TGF-β1+ siRNA-Hsp90-neg group and TGF-β1+ siRNA-Hsp90 group. Transwell invasion assay was used to investigate the effect of the interaction of Hsp90 and SIRT1 on the invasion ability of lung cancer A549 cells. The expressions of Hsp90, SIRT1, E-cadherin and vimentin were detected by Western blotting. The effect of inhibiting Hsp90 expression on the stability of SIRT1 protein and EMT of lung cancer A549 cells was observed.Results:After 48 h induction with TGF-β1, EMT characteristics of lung cancer A549 cells were induced successfully. The relative expression levels of Hsp90 protein in the control group and TGF-β1 group were 0.45±0.05 and 1.31±0.06, respectively, the relative expression levels of SIRT1 protein were 0.29±0.04 and 0.95±0.08, respectively, and there were statistically signigicant differences ( t=10.98, P=0.018; t=7.39, P=0.028). The results of immunocoprecipitation showed that there was an interaction between Hsp90 and SIRT1 protein in lung cancer A549 cells. The relative expression levels of Hsp90 in the TGF-β1 group, TGF-β1+ siRNA-Hsp90-neg group and TGF-β1+ siRNA-Hsp90 group were 0.75±0.07, 0.63±0.06 and 0.23±0.05, respectively, and there was a statistically significant difference ( F=18.85, P=0.012). The relative expression levels of SIRT1 in the above three groups were 0.99±0.08, 0.97±0.12 and 0.35±0.05, respectively, and there was a statistically significant difference ( F=16.52, P=0.014). The expression levels of Hsp90 and SIRT1 in the TGF-β1+ siRNA-Hsp90 group were significantly lower than those in the TGF-β1 group ( P=0.019, P=0.016). The numbers of cells passing Matrigel in the above three groups were 378.13±27.70, 323.52±19.82 and 142.51±22.54, respectively, and there was a statistically significant difference ( F=27.35, P=0.022). The number of cells passing Matrigel in the TGF-β1+ siRNA-Hsp90 group was significantly less than that in the TGF-β1 group ( P=0.028). The relative expression levels of E-cadherin in the above three groups were 0.31±0.02, 0.34±0.04 and 0.63±0.05, respectively, and there was a statistically significant difference ( F=19.39, P=0.031). The relative expression levels of vimentin in the above three groups were 0.33±0.02, 0.27±0.05 and 0.09±0.03, respectively, and there was a statistically significant difference ( F=12.58, P=0.012). The expression level of E-cadherin in the TGF-β1+ siRNA-Hsp90 group was significantly higher than that in the TGF-β1 group ( P=0.017), while the expression level of vimentin was significantly lower than that in the TGF-β1 group ( P=0.023). Conclusion:Hsp90 interacts with SIRT1, and Hsp90 inhibition can lead to the decrease of SIRT1 protein level. Hsp90 may play a role of molecular chaperone to maintain the conformation stability of SIRT1, and the interaction between Hsp90 and SIRT1 may be one of the molecular mechanisms for the occurrence of EMT and the enhancement of invasion ability of lung cancer A549 cells.