Wu Zhengrong, Li Gang, Wu Lirong, Weng Desheng, Li Xiangping, Yao Kaitai
1Department of Pathology & Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, PR China.
BMC Cancer. 2009 Sep 6;9:315. doi: 10.1186/1471-2407-9-315.
Human Cripto-1, a member of the EGF-CFC family, is indispensable for early embryonic development. Cripto-1 plays an important oncogenic role during tumorigenesis and is overexpressed in a wide range of epithelial carcinomas, yet little is known about Cripto-1 in nasopharyngeal carcinoma (NPC). The aim of this study was to analyze the roles of Cripto-1 in the progression and clinical characteristics in NPC clinical samples and cell lines.
The expression of Cripto-1 at mRNA level was detected by the reverse transcription-polymerase chain reaction (RT-PCR) and real time RT-PCR, and western blot was used to examine the protein expression. Cripto-1 expression and its clinical characteristics were investigated by performing immunohistochemical analysis on a total of 37 NPC clinical tissue samples. Lentiviral vectors were constructed to get an efficient expression of anti-Cripto-1 siRNA in CNE-2 and C666-1 cells, with invalid RNAi sequence as control. After the inhibition of the endogenous Cripto-1, the growth, cell cycle and invasion of cells were detected by MTT, FACS and Boyden chamber assay respectively. Moreover, in vivo, the proliferation of the tumor cells was evaluated in xenotransplant nude mice model with whole-body visualizing instrument.
The results of real-time RT-PCR and western blot showed that the expression level of Cripto-1 was markedly higher in NPC cell lines than that in the immortalized nasopharyngeal epithelial cell at both mRNA and protein levels. RT-PCR of 17 NPC tissues showed a high expression rate in 76.5% (13/17) cases. In an immunohistochemical study, Cripto-1 was found to express in 54.1% (20/37) cases of NPC. In addition, Cripto-1 overexpression was significantly associated with N classification (p = 0.034), distant metastasis (p = 0.036), and clinical stage (p = 0.007). Inhibition of endogenous Cripto-1 by lentivirus-mediated RNAi silencing technique suppressed NPC cell growth and invasion in vitro. In vivo, the average weight (p = 0.026) and volume (p = 0.044) of tumor in CNE-2/GFP+/Cripto-1- xenotransplant mice group were significantly lower than those in the control group. The Ki67 index was obviously lower in Cripto-1 RNAi treated tumors (p < 0.01).
Data of this study suggest that Cripto-1 overexpression is connected with the tumorigenesis and progression of NPC, lentivector-mediated RNAi might be feasible for the inhibition of the growth and invasion of NPC.
人CRIPTO-1是表皮生长因子- CFC家族成员之一，对早期胚胎发育至关重要。CRIPTO-1在肿瘤发生过程中发挥重要的致癌作用，在多种上皮癌中过表达，但关于其在鼻咽癌（NPC）中的研究甚少。本研究旨在分析CRIPTO-1在NPC临床样本和细胞系中的进展及临床特征中的作用。
采用逆转录-聚合酶链反应（RT-PCR）和实时RT-PCR检测CRIPTO-1在mRNA水平的表达，并用蛋白质印迹法检测蛋白质表达。通过对37例NPC临床组织样本进行免疫组织化学分析，研究CRIPTO-1表达及其临床特征。构建慢病毒载体，使抗CRIPTO-1 siRNA在CNE-2和C666-1细胞中高效表达，以无效RNAi序列作为对照。内源性CRIPTO-1被抑制后，分别通过MTT、流式细胞术和Boyden小室实验检测细胞的生长、细胞周期和侵袭能力。此外，在体内，用全身可视化仪器在异种移植裸鼠模型中评估肿瘤细胞的增殖情况。
实时RT-PCR和蛋白质印迹结果显示，NPC细胞系中CRIPTO-1在mRNA和蛋白质水平的表达均明显高于永生化鼻咽上皮细胞。17例NPC组织的RT-PCR结果显示，76.5%（13/17）的病例表达率较高。免疫组织化学研究发现，54.1%（20/37）的NPC病例中CRIPTO-1表达。此外，CRIPTO-1过表达与N分期（p = 0.034）、远处转移（p = 0.036）和临床分期（p = 0.007）显著相关。通过慢病毒介导RNAi沉默技术抑制内源性CRIPTO-1可在体外抑制NPC细胞生长和侵袭。在体内，CNE-2/GFP+/CRIPTO-1-异种移植小鼠组肿瘤的平均重量（p = 0.026）和体积（p = 0.044）明显低于对照组。在CRIPTO-1 RNAi处理的肿瘤中，Ki67指数明显较低（p < 0.01）。
本研究数据表明，CRIPTO-1过表达与NPC的肿瘤发生和进展有关，慢病毒介导RNAi可能对抑制NPC的生长和侵袭可行。
