自噬对口腔鳞癌上皮间充质转化的调节作用的观察

Regulation of autophagy on epithelial mesenchymal transformation in oral squamous cell carcinoma

目的:利用CQ(chloroquine,CQ)及雷帕霉素(rapamycin,RAPA)诱导Cal-27细胞,探讨自噬在口腔鳞癌上皮间充质转化(Epithelial mesenchymal transition,EMT)中的作用和机制。方法:应用TGF-β(transforming growth factor,TGF-β)诱导Cal-27细胞发生EMT。同时应用RAPA增强细胞自噬水平,应用CQ抑制细胞自噬水平。应用细胞划痕实验分布检测诱导后细胞迁移能力的改变;Transwell小室实验分别检测诱导后细胞的迁移能力。Western blot检测诱导3 d后ZO-1,vimentin,FN1等EMT相关蛋白水平的变化;统计软件进行分析。结果:以5 ng/mLTGF-β诱导Cal-27细胞3 d,E-cadherin明显下降,Vimentin明显上升,说明建立EMT模型成功。与空白组相比较,以5 ng/mLTGF-β诱导Cal-27细胞3 d后,划痕愈合率明显上升(P<0.05),穿模细胞数明显增多(P<0.05);继而分别以100 ng/mLRAPA和100 ng/mLCQ与5 ng/mLTGF-β共同诱导Cal-27细胞3 d,与TGF-β组相比较,RAPA共诱导组划痕愈合率明显下降(P<0.05),穿膜细胞数明显减少(P<0.05);与TGF-β组相比较,CQ共诱导组划痕愈合率明显升高(P<0.05),穿膜细胞数明显增多(P<0.05);与空白组相比较,以5 ng/mLTGF-β诱导Cal-27细胞3 d后,FN1、Vimentin表达量升高,ZO-1表达量降低;继而分别以100 ng/mL RAPA和100 ng/mLCQ与5 ng/mLTGF-β共同诱导Cal-27细胞3 d,与TGF-β组相比较,RAPA共诱导组FN1、Vimentin表达量降低,ZO-1表达量升高;与TGF-β组相比较,CQ共诱导组FN1、Vimentin表达量升高,ZO-1表达量降低。结论:TGF-β可以诱导Cal-27细胞建立EMT模型。在EMT模型中,促进自噬可以抑制EMT转化,抑制自噬可以促进EMT转化。

Objective: To investigate the role and mechanism of autophagy in epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma cells induced by CQ (chloroquine) and rapamycin (RAPA). Methods: TGF-β(transforming growth factor β) was used to induce EMT in Cal-27 cell line. At the same time, RAPA was used to enhance and CQ was used to inhibit autophagy. The ability of cell migration was detected by scratch distribution test and the ability of cell migration was detected by Transwell chamber test. Western blot was used to detect the changes of ZO-1, vimentin, FN1 and other EMT-related proteins after 3 days induction, and SPSS 22.0 statistical software was used to analyze the data. Results: After 3 days of induction with 5 ng/mL TGF-β, E-cadherin decreased significantly and Vimentin increased significantly. Compared with the control group, the wound healing rate increased significantly ( P <0.05) and the number of penetrating cells increased significantly ( P <0.05) after 3 days induction with 5 ng/mL TGF-β, and then the cells were co-induced with 100 ng/mL RAPA and 100 ng/mL CQ and 5 ng/mL TGF-β for 3 days. Compared with TGF-β group. The healing rate of the RAPA co-induced with 5 ng/mL TGF-β group decreased significantly ( P <0.05) and the number of penetrating cells decreased significantly ( P <0.05). Compared with TGF-β group. The healing rate of the CQ co-induced with 5 ng/mL TGF-β group increased significantly ( P < 0.05) and the number of penetrating cells increased significantly ( P <0.05). Compared with the control group, FN1 and Vimentin expression increased and ZO-1 expression decreased 3 days after induction with 5 ng/mL TGF-β. And then induced Cal-27 cells with 100 ng/mL RAPA and 100 ng/mL CQ and 5 ng/mL TGF-β respectively for 3 days. Compared with TGF-β group, FN1 and Vimentin expression decreased in RAPA co-induction group. Compared with TGF-β group, the expression of FN1 and Vimentin increased and the expression of ZO-1 decreased in CQ co-induction group. Conclusion: TGF-β can induce Cal-27 cells to establish EMT model. In EMT model, promoting autophagy can inhibit EMT , inhibiting autophagy can promote EMT.