Objective: Vascular endothelial growth factor A (VEGFA) was investigated as the key protein which might promote the specific metastasis progress of nasopharyngeal carcinoma. Methods: Sixteen specimens of pulmonary metastasis carcinoma and counterparts in primary nasopharyngeal carcinoma tissue were collected from patients. The expression of VEGFA through immunohistochemistry was investigated.VEGFA was knocked down by siRNA in two cell lines of nasopharyngeal carcinoma (CNE-1 and 5-8F), MTT and Transwell test were used to explore the role of VEGFA in praxiology. Then shRNA was used to cultivate the stable CNE-1 cell line with down-regulated-expression of VEGFA. The nude mice models were built through tail vein injection of specific nasopharyngeal carcinoma cells, and lungs were collected to perform further metastasis analysis. Results: Previous genetic studies showed that VEGFA had higher expression in metastasis tissue, and the result was validated in the present study using immunohistochemistry. The percentage of positive cells was 84.8% in pulmonary metastasis group, 51.5% in primary tissue group (t=8.639, P<0.05), average optical density was 0.154 in pulmonary metastasis group, 0.061 in primary tissue group (t=18.791, P<0.05). Low expression of VEGFA inhibited cell viability of optical density value of CNE-1 in siRNA gourp was 0.715, 0.902 in control group (t=7.274, P<0.05); 5-8F in siRNA group was 0.715, 0.935 in control group (t=7.751, P<0.05). Number counting suppressed migration of CNE-1 in siRNA group was 52 per high-power lens, 124 per high-power lens in control group (t=29.380, P<0.05), 5-8F in siRNA group was 65 per high-power lens, 155 per high-power lens in control group (t=18.181, P<0.05). Number counting invasion of CNE-1 in siRNA gourp was 38 per high-power lens, 86 per high-power lens in control group (t=27.665, P<0.05), 5-8F in siRNA group was 52 per high-power lens, 116 per high-power lens in control group (t=40.972, P<0.05) in vitro. Furthermore, knock-down of VEGFA in nasopharyngeal carcinoma reduced the pulmonary metastasis in vivo. Number counting of tumor volumes in shRNA group was 2.4, and 11.0 in control group (t=6.143, P<0.05); average optical density of immunohistochemistry in shRNA group was 0.033, and 0.176 in control group (t=15.734, P<0.05). Conclusions: Results above reveal the overexpression of VEGFA in nasopharyngeal carcinoma can facilitate the pulmonary metastasis. Targeting VEGFA may provide a new biomarker of clinical study.
目的： 研究促进鼻咽癌定向肺转移的蛋白位点，并对其在肿瘤发生发展中的作用进行验证。 方法： 收集16例患者鼻咽癌肺转移及对应原位鼻咽癌标本，通过免疫组织化学方法对血管内皮生长因子A(vascular endothelial growth factor A，VEGFA)的表达水平进行检测，比较VEGFA在鼻咽癌肺转移灶及原发灶中的表达差异。在鼻咽癌细胞株CNE-1和5-8F中，使用小分子干扰RNA(siRNA)转染抑制VEGFA表达，通过细胞迁移、细胞侵袭及四甲基偶氮唑蓝(MTT)实验比较VEGFA低表达与正常表达鼻咽癌细胞的功能差异，探究VEGFA对鼻咽癌细胞行为学的影响。其后在动物水平探讨VEGFA的作用，通过短发夹RNA(shRNA)抑制VEGFA，尾静脉注射入沉默组及对照组小鼠以构建裸鼠肺转移模型，通过比较两组小鼠肺部成瘤情况，验证鼻咽癌细胞VEGFA表达水平与鼻咽癌肺转移的关系。采用t检验进行统计学处理。 结果： VEGFA与小鼠鼻咽癌肺转移有关，鼻咽癌肺转移与原发灶临床标本的免疫组织化学染色涂片中阳性细胞数比例分别为84.8%和51.5%(t＝8.639，P<0.05)；平均吸光度为0.154和0.061(t＝18.791，P<0.05)。MTT实验证明VEGFA低表达可以抑制鼻咽癌细胞的活性，CNE-1细胞株中VEGFA低表达组与对照组的96 h吸光度分别为0.715和0.902(t＝7.274，P<0.05)；5-8F细胞株中VEGFA低表达组与对照组的96 h吸光度分别为0.715和0.935(t＝7.751，P<0.05)，Transwell实验证明抑制VEGFA可以下调鼻咽癌细胞的迁移能力，CNE-1细胞株中VEGFA低表达组与对照组的细胞计数分别为每高倍镜视野下52个和124个(t＝29.380，P<0.05)，5-8F中VEGFA低表达组与对照组的细胞计数分别为每高倍镜视野下65个和155个(t＝18.181，P<0.05)。同时可以抑制其侵袭能力，CNE-1细胞株中VEGFA低表达组与对照组的细胞计数分别为每高倍镜视野下38个和86个(t＝27.665，P<0.05)，5-8F中VEGFA低表达组与对照组的细胞计数分别为每高倍镜视野下52个和116个(t＝40.972，P<0.05)。小鼠实验证明敲低鼻咽癌细胞中VEGFA的表达可有效抑制鼻咽癌的肺转移，VEGFA抑制组与对照组的平均肺转移灶计数分别为2.4个和11.0个(t＝6.143，P<0.05)，免疫组化染色平均吸光度值分别为0.033和0.176(t＝15.734，P<0.05)。 结论： 鼻咽癌过表达VEGFA与其发生肺转移有关。VEGFA可能是潜在的临床预后评估标志。.
Keywords: Lung neoplasmas; Nasopharyngeal neoplasms; Neoplasmas metastasis; Vascular endothelial growth factor A.