阿帕替尼调控WWOX对子宫内膜癌细胞增殖、凋亡、迁移、侵袭的影响及其机制研究

目的:探讨阿帕替尼（Apatinib）是否通过包含氧化还原酶的WW结构域（WWOX）影响子宫内膜癌细胞增殖、凋亡、迁移、侵袭。方法:噻唑蓝（MTT）比色法检测4 μmol/L、8 μmol/L、16 μmol/L、32 μmol/L Apatinib作用于子宫内膜癌细胞HEC-1 24 h、48 h、72 h后的细胞活性，流式细胞术检测细胞凋亡率；蛋白质印迹法（Western blot）检测细胞周期蛋白D1（Cyclin D1）、p21、B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、基质金属蛋白酶-2（MMP-2）、E-钙黏蛋白（E-cadherin）、WWOX蛋白水平，Transwell小室法检测迁移细胞数、侵袭细胞数。在HEC-1中转染pcDNA3.1-WWOX，或转染si-WWOX并用16 μmol/L Apatinib进行处理，采用上述方法评估细胞增殖、凋亡、迁移、侵袭情况。结果:Apatinib明显降低HEC-1细胞活性（P＜0.05），呈剂量、时间依赖性；Apatinib显著增加HEC-1细胞凋亡率及p21、Bax蛋白表达量（P＜0.05），呈剂量依赖性；Apatinib明显降低Cyclin D1、Bcl-2蛋白表达量（P＜0.05），呈剂量依赖性；16 μmol/L Apatinib显著减少HEC-1细胞的迁移细胞数、侵袭细胞数、MMP-2蛋白表达量（P＜0.05），明显提高E-cadherin、WWOX蛋白表达量（P＜0.05）。过表达WWOX明显降低HEC-1细胞中Cyclin D1、Bcl-2、MMP-2蛋白表达量、细胞活性、迁移细胞数、侵袭细胞数（P＜0.05），提高p21、Bax、E-cadherin、WWOX蛋白表达量及细胞凋亡率（P＜0.05）。抑制WWOX表达逆转了Apatinib对HEC-1细胞中Cyclin D1、Bcl-2、MMP-2蛋白表达量、细胞活性、迁移、侵袭的抑制作用，以及逆转了其对p21、Bax、E-cadherin、WWOX蛋白表达量、细胞凋亡的促进作用。结论:阿帕替尼通过调控WWOX表达，抑制子宫内膜癌细胞增殖、迁移、侵袭，并诱导细胞凋亡。

Effect of Apatinib regulating WWOX on proliferation,apoptosis,migration and invasion of endometrial carcinoma cells and its mechanism

Objective:To investigate whether Apatinib affects proliferation,apoptosis,migration and invasion of endometrial cancer cells through WWOX.Methods:MTT assay was used to detect the activity of 4 μmol/L,8 μmol/L,16 μmol/L and 32 μmol/L Apatinib in endometrial cancer cells HEC-1 for 24 h,48 h and 72 h.Flow cytometry was applied to determine cells apoptosis rate.Western blot was employed to analyze the level of Cyclin D1,p21,Bcl-2,Bax,MMP-2,E-cadherin and WWOX protein,and the number of migrated cells and the number of invading cells were detected by Transwell chamber method.pcDNA3.1-WWOX was transfected into HEC-1,or si-WWOX was transfected and cells were treated with 16 μmol/L Apatinib,and cell proliferation,apoptosis,migration and invasion were evaluated by the above methods.Results:Apatinib significantly decreased the activity of HEC-1 cells in a dose- and time-dependent manner (P＜0.05).Apatinib obviously increased the HEC-1 cells apoptosis rate,p21 and Bax protein expression in a dose-dependent manner (P＜0.05).Apatinib greatly decreased Cyclin D1 and Bcl-2 protein level in a dose-dependent manner (P＜0.05).16 μmol/L Apatinib markedly reduced the number of migrated cells,invasive cells and MMP-2 protein expression in HEC-1 cells (P＜0.05),remarkably improved E-cadherin,WWOX protein level (P＜0.05).Overexpression of WWOX notably reduced Cyclin D1,Bcl-2,MMP-2 protein level in HEC-1 cells,cell viability,number of migrated cells,number of invasive cells (P＜0.05),while increased p21,Bax,E-cadherin,WWOX expression and apoptosis rate (P＜0.05).Inhibition of WWOX expression reversed the inhibitory effects of Apatinib on Cyclin D1,Bcl-2,MMP-2 protein expression,cell activity,migration and invasion in HEC-1 cells,and reversed the promotion of Apatinib on p21,Bax,E-cadherin,WWOX proteins expression and apoptosis.Conclusion:Apatinib inhibits the proliferation,migration,invasion and induces apoptosis of endometrial cancer cells by regulating WWOX expression.