摘要： 目的·探讨Ras相关结构域家族成员5（Ras association domain family 5，
RASSF5）对头颈鳞状细胞癌（head and neck squamous cell carcinoma，HNSCC）细胞侵袭和迁移能力的影响。方法·通过基因表达谱交互分析（Gene Expression Profiling Interactive Analysis，GEPIA）平台分析癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库中563例HNSCC的
RASSF5基因的表达情况及其对患者预后生存的影响。通过CMV-MCS-PGK-Puro载体构建
RASSF5过表达慢病毒（LV
RASSF5）和对照慢病毒（LV vector）。通过划痕实验、Transwell迁移和侵袭实验分析
RASSF5过表达对HNSCC细胞Cal27和HN30迁移和侵袭能力的影响；通过Western blotting分析
RASSF5过表达对上皮型钙黏蛋白（epithelial cadherin，E-cadherin）、Snail蛋白、糖原合酶激酶3β（glycogen synthase kinase 3β，GSK-3β）和磷酸化GSK-3β（phosphorylated GSK-3β，p-GSK-3β）的作用。构建BALB/c裸鼠肺转移模型，分别经尾静脉注射转染LV
RASSF5（
RASSF5过表达组）或LV vector（对照组）的Cal27细胞，每组5只小鼠；通过观察裸鼠肺部转移瘤结节数量分析过表达
RASSF5对Cal27细胞转移能力的影响。结果·
RASSF5基因在HNSCC中的表达水平低于正常组织（
P=0.001），低表达
RASSF5的患者总体生存期（
P=0.005）和无病生存期（
P=0.004）较差。划痕实验显示转染LV
RASSF5后，Cal27细胞和HN30细胞在观察终点的划痕面积高于相应的对照组细胞（
P=0.003，
P=0.015）。Transwell迁移实验显示转染LV
RASSF5后，Cal27细胞和HN30细胞穿出小室膜的数量低于相应对照组细胞（
P=0.005，
P=0.001）。Transwell侵袭实验同样显示转染LV
RASSF5的Cal27细胞和HN30细胞穿出预铺基质胶小室膜细胞数量低于对照组（
P=0.001，
P=0.001）。Western blotting结果显示过表达
RASSF5的Cal27和HN30细胞中，E-cadherin表达水平提高，而p-GSK-3β/GSK-3β比值以及Snail蛋白质水平降低。在裸鼠肺转移模型中，
RASSF5过表达组的肺部瘤结节数量少于对照组（
P=0.049）。结论·
RASSF5过表达抑制HNSCC细胞的侵袭和迁移能力，而通过GSK-3β/Snail信号通路抑制上皮间质转化可能在其中起到关键作用。
关键词: 头颈癌, Ras相关结构域家族成员5, 细胞迁移, 侵袭, 上皮间质转化 Abstract: Objective·To investigate the effect of Ras association domain family 5 (RASSF5) on migration and invasion of head and neck squamous cell carcinoma (HNSCC).
Methods·The expression of RASSF5 gene in 563 cases of HNSCC from the Cancer Genome Atlas (TCGA) database and the effect of RASSF5 expression on prognosis and survival of patients were analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) platform. The lentivirus overexpressing RASSF5 (LV RASSF5) and control lentivirus (LV vector) were constructed by CMV-MCS-PGK-Puro vector. The wound-healing assay and the Transwell assay were performed to test the invasion and metastasis capacity of overexpressed-RASSF5 HNSCC cells. In addition, the expressions of epithelial cadherin (E-cadherin), Snail, glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β (p-GSK-3β) were determined by Western blotting in RASSF5-overexpressed HNSCC cells. The lung metastasis model of HNSCC in 10 BALB/c nude mice were established. Cal27, which were transfected with LV RASSF5 or LV vector, were respectively injected into 5 BALB/c nude mice via tail vein, and the quantity of metastasis nodules was counted to evaluate the effect of RASSF5 overexpression on tumor metastasis ability.
Results·In HNSCC, the gene expression level of RASSF5 was lower than that in normal tissues (P=0.001). Patients with lower gene expression of RASSF5 had worse overall survival (P=0.005) and disease-free survival (P=0.004). The relative healing areas of Cal27 and HN30 transfected with LV RASSF5 were higher than those of the control group (transfected with LV vector) at the experimental endpoint of the wound-healing assay (P=0.015, P=0.003). The number of cells that had traversed the cell-permeable membrane was higher in Cal27 and HN30 cells transfected with LV RASSF5 than those in the control group in the Transwell migration assay (P=0.005, P=0.001). And in the Transwell invasion assay, the number of invaded cells that had traversed the matrigel-coated membrane was also higher in Cal27 and HN30 cells transfected with LV RASSF5 than those in the control group (P=0.001, P=0.001). Western blotting showed an increased level of E-cadherin, and decreased level of Snail and p-GSK-3β/GSK-3β ratio in RASSF5 overexpression Cal27 and HN30 cells. And in the metastasis assay of BALB/c nude mice, the quantity of metastases nodes was lower in the RASSF5-overexpressed group than that in the control group (P=0.049).
Conclusion·RASSF5 may inhibit epithelial-mesenchymal transition through GSK-3β/Snail pathway, and then inhibits the invasion and metastasis ability of HNSCC cells.
Key words: head and neck cancer, Ras association domain family 5 (RASSF5), cell migration, invasion, epithelial-mesenchymal transition PDF全文下载地址:
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