TGF-β1通过TRAF6活化Notch3信号通路促进卵巢癌细胞迁移和侵袭的研究

Transforming growth factor beta-1 promotes migration and invasion of ovarian epithelial carcinoma cells through TRAF6-mediated activation of Notch3 signaling pathway

背景与目的:转化生长因子β1(transforming growth factor-β1,TGF-β1)与Notch3在卵巢肿瘤组织中的异常表达与扩增分别与肿瘤转移和患者的低生存率有关。尽管TGF-β1与Notch3信号通路的交互作用促进多种肿瘤细胞的侵袭和转移,但其具体机制尚存在争议。该研究通过体外细胞学实验,探究TGF-β1与Notch3信号通路对卵巢癌细胞生物学行为的影响及其可能的相互作用机制。方法:以上皮性卵巢癌细胞Hey A8和Hey为细胞模型,用ELISA检测培养上清液中TGF-β1的表达;分别用500 ng/mL TGF-β1中和抗体(对照组)、10 ng/mL TGF-β1、50μmol/L DAPT、10 ng/mL TGF-β1和50μmol/L DAPT、50μmol/L TRAF6多肽抑制剂、10 ng/mL TGF-β1和50μmol/L TRAF6多肽抑制剂处理细胞,采用蛋白[质]印迹法(Western blot)检测各处理组TGF-β1和Notch3信号通路分子以及TRAF6蛋白表达水平的变化;采用细胞计数试剂盒(cell counting kit-8,CCK-8)、划痕实验和Transwell小室测定各处理组细胞增殖、迁移和侵袭能力的变化。结果:Hey A8和Hey细胞培养上清液中TGF-β1浓度随时间延长而明显增加;与对照组相比,TGF-β1处理组Notch3-ICD、Hes1表达增加,Notch3抑制剂DAPT与TGF-β1共同处理组Notch3-ICD、Hes1表达水平无明显变化,TGF-β1明显促进细胞增殖、迁移和侵袭,Notch3抑制剂DAPT削弱了TGF-β1对卵巢癌细胞的促增殖、迁移和侵袭能力,说明TGF-β1通过激活Notch3信号通路促进卵巢癌细胞增殖、迁移和侵袭;进一步研究发现,TGF-β1激活Notch3信号通路的同时上调TRAF6表达,特异性抑制TRAF6能够抑制TGF-β1对Notch3信号通路的激活作用。结论:TGF-β1可能通过TRAF6活化Notch3信号通路,从而促进卵巢癌细胞的增殖、迁移和侵袭能力。

Background and purpose： Abnormal expression and amplification of transforming growth factor beta 1 （TGF-β1） and Notch3 in ovarian carcinoma tissues are associated with metastasis and low survival rate, respectively. The crosstalk between TGF-β1 and Notch3 signaling pathway promotes invasion and metastasis in various cancers. However, the mechanism is still under debate. Therefore, this study was designed, using in vitro cytological assays, to investigate the effects of TGF-β1 and Notch3 signaling pathway on ovarian cancer cell biological behavior and the potential mechanisms in terms of the crosstalk between TGF-β1 and Notch3 signaling pathway. Methods： Hey A8 and Hey cell lines were used as models in the study. The levels of TGF-β1 in supernatants from culture media were measured by ELISA. Both cell lines were treated with 500 ng/mL TGF-β1 neutralizing antibody （control group）, 10 ng/mL TGF-β1, 50 μmol/L DAPT, 10 ng/mL TGF-β1 and 50 μmol/L DAPT, 50 μmol/L tumor necrosis factor receptor-associated factor 6 （TRAF6） peptide inhibitor, 10 ng/mL TGF-β1 and 50 μmol/L TRAF6 peptide inhibitor, respectively. The protein expression levels of TGF-β1 and Notch3 signaling pathway molecules as well as TRAF6 from cell lines with different treatments were detected by Western blot. Cell proliferation, migration and invasion were tested by cell counting kit-8 （CCK-8）, scratch and Transwell assays, respectively. Results： The levels of TGF-β1 were timedependently increased in supernatants of culture media from Hey A8 and Hey cell lines. Compared with control group, TGF-β1 treatment increased the expression levels of Notch3-ICD and Hes1, while no obvious change was observed in the group treated with DAPT and TGF-β1. Moreover, TGF-β1 promoted cell proliferation, migration and invasion while DAPT decreased the proliferation, migration and invasion in cell lines treated with TGF-β1. These results indicated that TGF-β1 might promote proliferation, invasion and migration of ovarian epithelial cancer cells through activating the Notch3 signaling pathway. Further study showed that TGF-β1 up-regulated TRAF6 and activated the Notch3 signaling pathway. The activation of the Notch3 signaling pathway by TGF-β1 was inhibited in cells treated with the TRAF6 specific inhibitor. Conclusions： TGF-β1 may promote the proliferation, invasion and migration of ovarian epithelial carcinoma cells through TRAF6-mediated activation of the Notch3 signaling pathway.