Tie2在口腔鳞状细胞癌中的表达及其对细胞增殖、迁移及上皮-间充质转化过程的影响

Expression of Tyrosine Kinase Receptor 2 in Oral Squamous Cell Carcinoma and the Effect on Cell Proliferation and Migration and Epithelial-Mesenchymal Transition Process

目的 研究酪氨酸激酶受体2(tyrosine kinase receptor 2, Tie2)在口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)中的表达情况及其对细胞增殖、迁移及上皮-间充质转化(epithelial-mesenchymal transition, EMT)过程的影响。方法 采用免疫组织化学(immunohistochemistry, IHC)检测Tie2在OSCC组织与正常口腔黏膜组织中的表达。Western blot检测DOK细胞及OSCC细胞株中Tie2的表达,选取Tie2高表达细胞株作为实验株。将沉默Tie2的慢病毒载体成功转染至实验株用于后续实验。CCK-8及克隆形成实验检测细胞增殖、克隆能力;细胞划痕和Transwell实验检测细胞迁移能力;激光共聚焦显微镜检测细胞骨架纤丝状肌动蛋白(F-actin)重塑能力及上皮钙黏素(E-cadherin)、神经钙黏素(Ncadherin)的表达;Western blot检测EMT相关标志性蛋白E-cadherin、N-cadherin、波形蛋白(vimentin)的表达以及蛋白激酶B(protein kinase B, AKT)、胞外信号调节激酶(extracellular signal-regulated kinase, ERK)的表达。结果 IHC结果显示:OSCC组Tie2阳性率(74.5%)高于对照组(19.4%)(P<0.000 1)。与DOK细胞相比,Tie2在HSC-4及SCC-9细胞株呈现高表达,慢病毒shRNA-162组沉默效果最好,作为实验组并应用于后续实验。与对照组相比,实验组细胞的增殖、克隆及迁移能力明显降低,细胞骨架F-actin绿色荧光强度降低,细胞前缘丝状伪足数量减少、长度变短;E-cadherin的表达明显增加,Ncadherin、vimentin的表达明显降低。p-AKT及p-ERK蛋白表达降低,AKT及ERK蛋白的表达增加。结论 Tie2在大多数OSCC中呈高表达,沉默Tie2能通过调控AKT及ERK信号通路抑制OSCC细胞增殖克隆迁移能力,抑制F-actin重塑,改变其EMT相关标志性蛋白的表达,提示Tie2可能参与了OSCC的生长、转移及EMT过程。

Objective To study the expression of tyrosine kinase receptor 2(Tie2) in oral squamous cell carcinoma(OSCC) and its effect on cell proliferation and migration and the epithelial-mesenchymal transition(EMT)process. Methods Immunohistochemistry(IHC) tests were conducted to examine the expression of Tie2 in OSCC tissues and normal oral mucosa tissues. Western blot was performed to examine the expression of Tie2 in dysplastic oral keratinocyte(DOK) cell line and OSCC cell lines, and the cell line with high Tie2 expression was selected as the experimental cell line. The Tie2-silenced lentiviral vector was successfully transfected onto the experimental cell line for subsequent experiments. Cell proliferation and cloning abilities were examined with CCK-8 and clone formation assays.Cell migration ability was examined with scratch and Transwell assays. The remodeling ability of cytoskeletal F-actin and the expressions of E-cadherin and N-cadherin were examined with confocal laser scanning microscope. Western blot was performed to examine the expression of EMT-related signature proteins, including E-cadherin, N-cadherin, and vimentin,and the expression of the protein kinase B(AKT) and extracellular signal-regulated kinase(ERK). Results IHC results showed that the Tie2-positive rate of the OSCC group(74.5%) was significantly higher than that of the control group(19.4%)(P<0.000 1). The expression of Tie2 was higher in HSC-4 and SCC-9 cell lines compared to that in DOK cells. The lentiviral shRNA-162 group showed the best silencing effect, which was used as the experimental group and applied in subsequent experiments. Compared with those of the control group, the proliferation, cloning and migration capacities of the cells of the experimental group were significantly reduced. Furthermore, the green fluorescence intensity of the cytoskeleton F-actin was reduced, the number of filamentous pseudopods at the leading edge of the cells decreased and their length was shortened, and the expression of E-cadherin was significantly increased, while the expression of Ncadherin and vimentin was significantly reduced in the experimental group in comparison with those of the control group. The expression of p-AKT and p-ERK proteins decreased, while AKT and ERK protein expression increased.Conclusion Tie2 was highly expressed in most OSCC cells. Silencing Tie2 can inhibit the proliferation, cloning, and migration ability of OSCC cells, inhibit F-actin remodeling, and alter the expression of its EMT-related signature proteins by regulating AKT and ERK signaling pathway, which suggests that Tie2 may be involved in the growth, metastasis and EMT process of OSCC.