Hsp90与SIRT1相互作用对肺癌细胞发生EMT的调控作用

doi:10.3760/cma.j.cn371439-20201228-00040

摘要/Abstract

摘要：

目的探讨热休克蛋白90(Hsp90)与沉默信息调节因子2同源蛋白1(SIRT1)的相互作用及其对肺癌A549细胞发生上皮-间质转化(EMT)的影响。方法采用5 μg/L转化生长因子-β1(TGF-β1)作用于肺癌A549细胞建立EMT模型,分为对照组和TGF-β1组;采用免疫共沉淀法检测肺癌A549细胞中Hsp90与SIRT1的相互作用;采用RNA干扰技术沉默Hsp90基因的表达,分为TGF-β1组、TGF-β1+siRNA-Hsp90-neg组、TGF-β1+siRNA-Hsp90组;采用Transwell侵袭实验研究Hsp90与SIRT1的相互作用对肺癌A549细胞侵袭能力的影响;采用蛋白质印迹法分别检测Hsp90、SIRT1、上皮钙黏素以及波形蛋白的表达情况,观察抑制Hsp90表达对SIRT1蛋白稳定性及肺癌A549细胞EMT的影响。结果采用TGF-β1诱导48 h后,肺癌A549细胞呈现EMT变化,对照组和TGF-β1组Hsp90蛋白的相对表达量分别为0.45±0.05、1.31±0.06,SIRT1蛋白的相对表达量分别为0.29±0.04、0.95±0.08,差异有统计学意义(t=10.98,P=0.018;t=7.39,P=0.028)。免疫共沉淀结果显示,肺癌A549细胞中Hsp90和SIRT1蛋白之间存在相互作用;TGF-β1组、TGF-β1+siRNA-Hsp90-neg组、TGF-β1+siRNA-Hsp90组Hsp90蛋白的相对表达量分别为0.75±0.07、0.63±0.06、0.23±0.05,差异具有统计学意义(F=18.85,P=0.012),上述3组SIRT1蛋白的相对表达量分别为0.99±0.08、0.97±0.12、0.35±0.05,差异具有统计学意义(F=16.52,P=0.014),TGF-β1+siRNA-Hsp90组细胞中Hsp90和SIRT1表达水平均显著低于TGF-β1组(P=0.019,P=0.016)。上述3组通过Matrigel基质胶的细胞数量分别为378.13±27.70、323.52±19.82、142.51±22.54,差异具有统计学意义(F=27.35,P=0.022),TGF-β1+siRNA-Hsp90组通过Matrigel基质胶的细胞数量明显少于TGF-β1组(P=0.028)。上述3组上皮钙黏素蛋白相对表达量分别为0.31±0.02、0.34±0.04、0.63±0.05,差异具有统计学意义(F=19.39,P=0.031),波形蛋白相对表达量分别为0.33±0.02、0.27±0.05、0.09±0.03,组间差异具有统计学意义(F=12.58,P=0.012),TGF-β1+siRNA-Hsp90组细胞中上皮钙黏素表达水平显著高于TGF-β1组(P=0.017),而波形蛋白表达水平显著低于TGF-β1组(P=0.023)。结论 Hsp90与SIRT1存在相互作用,抑制Hsp90表达可导致SIRT1蛋白水平降低,Hsp90可能发挥分子伴侣功能来维持SIRT1蛋白构象稳定,Hsp90与SIRT1的相互作用可能是肺癌A549细胞发生EMT并增强侵袭能力的分子机制之一。

关键词: 癌, 非小细胞肺, 上皮-间质转化, 蛋白质相互作用图, 热休克蛋白质类, SIRT1

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.

Key words: Carcinoma, non-small-cell lung, Epithelial-mesenchymal transition, Protein interaction maps, Heat-shock proteins, SIRT1

侯江厚, 姚颖杰, 詹晓燕, 杨奕梅. Hsp90与SIRT1相互作用对肺癌细胞发生EMT的调控作用[J]. 国际肿瘤学杂志, 2021, 48(4): 200-205.

Hou Jianghou, Yao Yingjie, Zhan Xiaoyan, Yang Yimei. Regulation of EMT in lung cancer cells by interaction of Hsp90 and SIRT1[J]. Journal of International Oncology, 2021, 48(4): 200-205.

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