Cripto-1 overexpression is involved in the tumorigenesis of nasopharyngeal carcinoma.
BACKGROUND
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Wu
Zhengrong
Z
1Department of Pathology & Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, PR China. wuzr@yahoo.cn
Li
Gang
G
Wu
Lirong
L
Weng
Desheng
D
Li
Xiangping
X
Yao
Kaitai
K
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D002277:Carcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism; D005260:Female; D058851:GPI-Linked Proteins; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008875:Middle Aged; D009303:Nasopharyngeal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009368:Neoplasm Transplantation; D009385:Neoplastic Processes
D058851:GPI-Linked Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C070636:TDGF1 protein, human; D004815:Epidermal Growth Factor