摘要
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
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)
作者
张键
康敏
ZHANG Jian;KANG Min(Department of Gastroenterology,Affiliated Hospital of Southwest Medical University,Sichuan Luzhou 646000,China)
出处
《现代肿瘤医学》
CAS
北大核心
2022年第24期4445-4453,共9页
Journal of Modern Oncology
基金
西南医科大学附属医院博士科研启动基金资助项目(编号:21037)。
