AQP3在胰腺癌中表达及其在EGF诱导下对PANC-1细胞增殖迁移作用的实验研究
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摘要
目的：检测水通道蛋白3(aquaporins-3,AQP3)在胰腺癌组织和胰腺正常组织中的表达及其临床意义,初步探讨表皮生长因子(epidermal growth factor, EGF)通过相关信号通路上调AQP3对胰腺癌细胞增殖和迁移力的影响。
方法：取胰腺癌手术切除的组织标本36例,分别应用逆转录聚合酶链反应(RT-PCR)、免疫组化(Immunohistochemistry)、免疫荧光(immunofluorescence)和免疫印迹方法(Western-blot)检测胰腺组织和胰腺正常组织中AQP3表达与分布情况。在不同浓度和时间条件下观察EGF处理胰腺癌PANC-1细胞系AQP3表达水平,MTT和细胞迁移实验(phagokinetic track motility)测定法分别检测细胞的增殖和迁移能力。
结果：通过RT-PCR检测发现,AQP3mRNA在胰腺癌组织表达丰富,胰腺正常组织中无或低水平表达,存在明显差异(两者P<0.05),免疫组织化学结果显示AQP3阳性反应呈棕黄色,主要存在于细胞膜；免疫荧光试验显示同样的结果。通过Western-blot检测进一步证实了RT-PCR的结果。分析胰腺癌组织中AQP3表达量与患者的临床病理特征之间的关系,结果发现低分化腺癌AQP3的表达量明显高于高分化腺癌,与肿瘤的恶性度呈正相关。EGF可诱导表皮生长因子受体(epidermal growth factor receptor, EGFR)和细胞外信号调节激酶(ERK)磷酸化,EGF处理后5min,EGFR和ERK的磷酸化达到峰值,EGFR激酶抑制剂(PD153035)和ERK抑制剂(U0126)可阻断EGF诱导的ERK磷酸化；EGF可显著提高细胞中AQP3的活性,并随时间延长活性增强,24h达到较高水平,AQP3活性亦随浓度升高而增强,当EGF浓度为100gg/L时达到较高水平：PD153035、U0126和CuSO4可抑制AQP3的活性以及细胞的移行。
结论：AQP3在胰腺癌组织和胰腺正常组织之间存在差异表达；低分化腺癌AQP3的表达量明显高于高分化腺癌,AQP3的表达量与肿瘤的恶性度呈正相关。在胰腺癌PANC-1细胞中,EGF通过磷酸化EGFR,激活ERK,进而使AQP3表达上调,促进PANC-1细胞增殖和迁移。这一通路可被EGFR激酶抑制剂、ERK和AQP3抑制剂阻断,这些涉及的信号通路可能形成潜在的治疗胰腺癌的靶点。
Objective:The aim of this study was to investigate whether AQP3expression in the human pancreatic cancer cell lines, PANC-1,enhances cell migration. In this study we investigated whether AQP3is expressed in cultured PANC-1pancreatic cancer cells, and whether EGF induces AQP3expression via ERK signal transduction pathways and further more enhances cell migration.
Methods:The expression of AQP3in mRNA level was detected in pancreatic cancer cell lines PANC-1and pancreatic cancer cell line PANC-1by RT-PCR. We screened the expression profile of AQP3in pancreatic cancer tissues and corresponding normal mucosa from80patients with pancreatic cancer by RT-PCR, western blot analysis, immunochemical assay and immunofluorescence. The relationship between AQP3expression and clinicopathologic characteristics of patients was evaluated. Cultured PANC-1cells were treated with epidermal growth factor (EGF) and subjected to cell migration assay. The expression or activation level of proteins was analyzed by western blot.
Results:Here, we showed that AQP3is expressed in the human pancreatic cancer cell lines PANC-1. The EGF induced AQP3expression in a time-and dose-dependent manner and increased pancreatic cancer cell migration. Further more, a mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126inhibited EGF-induced AQP3expression and cell migration.
Conclusions:AQP3plays a critical role in EGF-induced cancer cell migration and that EGF induces AQP3expression via ERK signal transduction pathways.These finds provide evidence for a novel role of AQP3in pancreatic cancer as a potentially important determinant of tumor growth and spread. EGFR/ERK pathway mediated AQP3activation and cell migration stimulated by EGF in cultured PANC-1pancreatic cancer cell in vitro, and this cell signaling pathway can be inhibited by EGFR inhibitor and ERK inhibitor, which may be used as potential therapeutic targets in the treatment of pancreatic cancer.
方法：取胰腺癌手术切除的组织标本36例,分别应用逆转录聚合酶链反应(RT-PCR)、免疫组化(Immunohistochemistry)、免疫荧光(immunofluorescence)和免疫印迹方法(Western-blot)检测胰腺组织和胰腺正常组织中AQP3表达与分布情况。在不同浓度和时间条件下观察EGF处理胰腺癌PANC-1细胞系AQP3表达水平,MTT和细胞迁移实验(phagokinetic track motility)测定法分别检测细胞的增殖和迁移能力。
结果：通过RT-PCR检测发现,AQP3mRNA在胰腺癌组织表达丰富,胰腺正常组织中无或低水平表达,存在明显差异(两者P<0.05),免疫组织化学结果显示AQP3阳性反应呈棕黄色,主要存在于细胞膜；免疫荧光试验显示同样的结果。通过Western-blot检测进一步证实了RT-PCR的结果。分析胰腺癌组织中AQP3表达量与患者的临床病理特征之间的关系,结果发现低分化腺癌AQP3的表达量明显高于高分化腺癌,与肿瘤的恶性度呈正相关。EGF可诱导表皮生长因子受体(epidermal growth factor receptor, EGFR)和细胞外信号调节激酶(ERK)磷酸化,EGF处理后5min,EGFR和ERK的磷酸化达到峰值,EGFR激酶抑制剂(PD153035)和ERK抑制剂(U0126)可阻断EGF诱导的ERK磷酸化；EGF可显著提高细胞中AQP3的活性,并随时间延长活性增强,24h达到较高水平,AQP3活性亦随浓度升高而增强,当EGF浓度为100gg/L时达到较高水平：PD153035、U0126和CuSO4可抑制AQP3的活性以及细胞的移行。
结论：AQP3在胰腺癌组织和胰腺正常组织之间存在差异表达；低分化腺癌AQP3的表达量明显高于高分化腺癌,AQP3的表达量与肿瘤的恶性度呈正相关。在胰腺癌PANC-1细胞中,EGF通过磷酸化EGFR,激活ERK,进而使AQP3表达上调,促进PANC-1细胞增殖和迁移。这一通路可被EGFR激酶抑制剂、ERK和AQP3抑制剂阻断,这些涉及的信号通路可能形成潜在的治疗胰腺癌的靶点。
Objective:The aim of this study was to investigate whether AQP3expression in the human pancreatic cancer cell lines, PANC-1,enhances cell migration. In this study we investigated whether AQP3is expressed in cultured PANC-1pancreatic cancer cells, and whether EGF induces AQP3expression via ERK signal transduction pathways and further more enhances cell migration.
Methods:The expression of AQP3in mRNA level was detected in pancreatic cancer cell lines PANC-1and pancreatic cancer cell line PANC-1by RT-PCR. We screened the expression profile of AQP3in pancreatic cancer tissues and corresponding normal mucosa from80patients with pancreatic cancer by RT-PCR, western blot analysis, immunochemical assay and immunofluorescence. The relationship between AQP3expression and clinicopathologic characteristics of patients was evaluated. Cultured PANC-1cells were treated with epidermal growth factor (EGF) and subjected to cell migration assay. The expression or activation level of proteins was analyzed by western blot.
Results:Here, we showed that AQP3is expressed in the human pancreatic cancer cell lines PANC-1. The EGF induced AQP3expression in a time-and dose-dependent manner and increased pancreatic cancer cell migration. Further more, a mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126inhibited EGF-induced AQP3expression and cell migration.
Conclusions:AQP3plays a critical role in EGF-induced cancer cell migration and that EGF induces AQP3expression via ERK signal transduction pathways.These finds provide evidence for a novel role of AQP3in pancreatic cancer as a potentially important determinant of tumor growth and spread. EGFR/ERK pathway mediated AQP3activation and cell migration stimulated by EGF in cultured PANC-1pancreatic cancer cell in vitro, and this cell signaling pathway can be inhibited by EGFR inhibitor and ERK inhibitor, which may be used as potential therapeutic targets in the treatment of pancreatic cancer.
引文
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