血管生成素2异常表达与肺癌侵袭、转移和预后的关系

Abnormal expression of angiopoietin-2 associated with invasion,metastasis and prognosis of lung cancer

目的探讨血管生成素2（Ang－2）异常表达与肺癌侵袭、转移和预后的关系。 方法以自身配对法收集南通大学附属医院2009年1月至2010年12月间122例原发性肺癌患者术后癌和癌旁组织，以免疫组织化学法分析组织Ang－2表达。在细胞水平上，观察肺上皮Beas－2B及肺癌（SPCA－1，NCI－1650，A549和NCI－H1975）细胞中Ang－2蛋白及mRNA表达。筛选干扰Ang－2基因有效Short hairpin RNA（shRNA）质粒转染高表达Ang－2的A549肺癌细胞，以Western印迹、荧光定量聚合酶链反应、细胞计数试剂盒8（CCK－8）、Transwell等观察转染前后肺癌细胞Ang－2表达、Ang－2 mRNA水平、细胞增殖、侵袭、迁移及上皮间质转化（EMT）能力等的变化，分析Ang－2表达与肺癌侵袭及转移的关系。 结果肺癌组织Ang－2高表达，Ang－2表达与肿瘤直径（P＝0.008）、分化程度（P＝0.033）、TNM分期（P＝0.025）和5年生存率（P〈0.001）均显著相关。Ang－2高表达者5年生存率显著低于低表达者（16.1%比80.0%，P〈0.001）；TNM分期Ⅰ期患者Ang－2高表达者5年生存率显著低于低表达者（P〈0.001），Ⅱ、Ⅲ和Ⅳ期患者Ang－2高、低表达者之间5年生存率差异均无统计学意义。Ang－2蛋白及mRNA在肺癌细胞A549中表达最高。以最高效的Ang－2－shRNA－1质粒转染A549细胞后，细胞中Ang－2表达明显受抑，细胞增殖速度明显降低并呈时间依赖性；细胞的侵袭、迁移及EMT能力亦显著下降。 结论Ang－2异常表达与肺癌侵袭、转移及患者预后密切相关，干扰Ang－2表达有望成为肺癌治疗新的分子靶目标。

ObjectiveTo investigate the relationship between abnormal angiopoietin-2（Ang-2）expression and invasion/metastasis of lung cancer. MethodsTotally 122 cases of postoperative primary lung cancer tissues and their paracancerous tissues from Jan.2009 to Dec.2010 were collected from Affiliated Hospital of Nantong University and Ang-2 expression was analyzed by immunohistochemistry.At the cellular level,the protein and mRNA levels of Ang-2 in lung epithelial cell line Beas-2B and four lung cancer cell lines（SPCA-1,NCI-1650,A549 and NCI-H1975）were observed.The most effective Ang-2-shRNA for Ang-2 transcription was screened and transfected into A 549 lung cancer cells.The Ang-2 expression,Ang-2 gene transcription,cell proliferation,invasion/metastasis,and epithelial-mesenchymal transition（EMT）abilities of lung cancer cells were analyzed by Western blotting,fluorescent quantitative reverse transcriptase PCR,Cell Counting Kit-8 assay,and Transwell cell models for exploring the relationship between Ang-2 expression and invasion/metastasis of lung cancer. ResultsThe higher Ang-2 expression levels in lung cancerous tissues were closely related to tumor diameter（P=0.008）,differentiating degree（P=0.033）,TNM stage（P=0.025）and 5-year survival rate（P〈0.001）.According to the Kaplan-Meier survival curves,the 5-year survival rate of patients with higher expression levels of Ang-2（16.1%）was significantly poorer than that of patients with lower Ang-2（80.0%,P〈0.001）.Significant difference of 5-year survival rate was found in patients with different Ang-2 levels at TNM stage Ⅰ（P〈0.001）,but not at stage Ⅱ,Ⅲ and Ⅳ.Among Beas-2B and four lung cancer cell lines,the protein and mRNA levels of Ang-2 in A549 cells were the highest.After Ang-2-shRNA-1 plasmid successfully transfected into A549 cells,cell proliferation rate was significantly lower than that in the shRNA-negative or blank group at a time-dependent manner.The significant decrease of the invasion,migration and EMT abilities were also found in A549 cells after transfection of Ang-2 shRNA. ConclusionAbnormal expression of Ang-2 is closely related to invasion,migration and prognosis of lung cancer,and interfering the activation of Ang-2 would be a novel molecular-targeted therapy for lung cancer.