EphB1通过PI3K/AKT信号通路促进食管鳞状细胞癌EC-9706细胞增殖、迁移、侵袭并抑制其凋亡

目的:研究促红细胞生成素产肝细胞受体B1(erythropoietin-producing human hepatocellular receptor B1，EphB1)对食管鳞状细胞癌EC-9706细胞增殖、迁移、侵袭及凋亡的影响，并探索其可能的作用机制。方法:qRT-PCR法分析40对食管鳞状细胞癌手术患者的癌组织及邻近正常组织中EphB1表达情况，并分析其临床特征。在EC-9706细胞中分别转染si-EphB1 RNA和oe-EphB1 RNA及其阴性对照。通过qRT-PCR和蛋白免疫印迹实验检测转染效率；CCK-8实验分析EphB1对细胞活力的影响；Hoechst 33258染色和流式细胞学实验检测细胞凋亡；划痕愈合实验及Transwell侵袭实验明确细胞迁移及侵袭能力；蛋白免疫印迹实验检测EphB1蛋白、增殖相关蛋白［增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）］、凋亡相关蛋白［Bad、Bcl-2、Caspase 3 及剪切型-Caspase 3（cleaved-Caspase 3）］、侵袭转移相关蛋白［基质金属蛋白酶9(matrix metalloproteinase-9，MMP-9)、基质金属蛋白酶2(matrix metalloproteinase-2，MMP-2）、Snail、波形蛋白(Vimentin)、N-cadherin、E-cadherin］以及PI3K/AKT信号通路相关蛋白（PI3K、AKT、p-AKT）的表达水平。结果:EphB1在食管鳞状细胞癌组织及细胞系中高表达(P均＜0.05)，EphB1的表达水平与食管鳞状细胞癌患者的TNM分期、有无淋巴结转移和远处转移具有显著相关性(P均＜0.05)。与对照组相比，通过细胞转染技术沉默EphB1和过表达EphB1使EC-9706细胞的增殖、迁移、侵袭能力明显降低或增强，并诱导或抑制细胞凋亡(P均＜0.05)。在蛋白水平上，si-EphB1和oe-EphB1处理EC-9706细胞48 h后，增殖相关蛋白PCNA蛋白表达水平分别显著降低或增加（P均＜0.05），并分别增加或降低促凋亡相关蛋白Bad、cleaved-Caspase 3的表达水平（P均＜0.05），下调或上调抗凋亡蛋白Bcl-2、Caspase 3的蛋白表达（P均＜0.05），抑制或促进侵袭转移相关蛋白MMP-9、MMP-2、Snail、Vimentin、N-cadherin的蛋白活性，但却上调或下调 E-cadherin的表达水平（P均＜0.05）。同时，Western blotting实验结果还证实EphB1可诱导食管鳞状细胞癌EC-9706细胞中通路蛋白PI3K、p-AKT的活性增强（P均＜0.05）。结论:EphB1可能通过调控PI3K/AKT信号通路促进食管鳞状细胞癌EC-9706细胞的增殖、迁移及侵袭，并抑制其凋亡。

EphB1 promotes the proliferation,migration,invasion and inhibits the apoptosis of esophageal squamous cell carcinoma EC-9706 cells via the PI3K/AKT signaling pathway

Objective:To investigation the effects of erythropoietin-producing human hepatocellular receptor B1 (EphB1) on proliferation,migration,invasion and apoptosis in esophageal squamous cell carcinoma EC-9706 cells and explore the potential mechanism.Methods:qRT-PCR detected EphB1 expression in the cancer tissues and adjacent normal tissues of the 40 cases of esophageal squamous cell carcinoma patients underwent surgery,and the clinical characteristics were analyzed.Ec-9706 cells were transfected with si-EphB1 RNA,oe-EphB1 RNA and negetive control,respectively.qRT-PCR assay and Western blotting assay detected the transfection efficiency.The effects of EphB1 on cell viability were analyzed by CCK-8 assay.Hoechst 33258 staining and flow cytometry assays were used to detect cells apoptosis.Wound healing assay and Transwell invasion assay analysed the migration and invasion abilities of EC-9706 cells.The expression levels of EphB1 protein,proliferation related protein ［proliferating cell nuclear antigen (PCNA)］,apoptosis related proteins (Bad,Bcl-2,Caspase 3,cleaved-Caspase 3),invasion and metastasis related proteins ［matrix metalloproteinase-9 (MMP-9),matrix metalloproteinase-2 (MMP-2),Snail,Vimentin,N-cadherin,E-cadherin］ and PI3K/AKT signaling pathway related proteins (PI3K,AKT,p-AKT) were detected by Western blotting assay.Results:EphB1 was highly expressed in esophageal squamous cell carcinoma tissues and cell lines (P＜0.05),and expression level of EphB1 was significantly correlated with TNM stage,lymph node metastasis and distant metastasis in esophageal squamous cell carcinoma patients (P＜0.05).Silencing EphB1 or overexpression of EphB1 by cell transfection technique significantly reduced or enhanced the proliferation,migration,and invasion abilities of EC-9706 cells,and induced or inhibited cells apoptosis compared with the control cells (P＜0.05).At the protein level,treatment of EC-9706 cells with si-EphB1 and oe-EphB1 for 48 h obviously decreased or increased the expression levels of proliferation related protein PCNA (P＜0.05),increased or decreased the expression levels of pro-apoptotic related proteins Bad and cleaved-Caspase 3 (P＜0.05),downregulated or upregulated the expression levels of anti-apoptotic proteins Bcl-2 and Caspase 3 (P＜0.05),and inhibited or increased the expression levels of invasion and metastasis related proteins MMP-9,MMP-2,Snail,Vimentin and N-cadherin (P＜0.05),upregulated or downregulated the expression levels of E-cadherin (P＜0.05),respectively.Western blotting analysis showed that EphB1 induced the increase of PI3K,p-AKT protein activity in EC-9706 cells (P＜0.05).Conclusion:EphB1 may promote the proliferation,migration,invasion,and inhibit apoptosis of esophageal squamous cell carcinoma EC-9706 cells by the activation of the PI3K/AKT signaling pathway.