[Influence of suppression of Epstein-Barr Virus-encoded latent membrane protein 1 by rAAV vector mediated RNA interference on metastatic ability of nasopharyngeal cancer cells in vivo].
OBJECTIVE
To study the effect of Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) on the tumor growth and metastasis of nasopharyngeal carcinoma (NPC) cells in vivo and its possible mechanism.


METHODS
To construct two recombinant adeno-associated virus (rAAV): rAAV-shRNA-LMP-1 and rAAV-EGFP (enhanced green fluorescent protein). Multiplicity of infection (MOI) was confirmed by using different titre of rAAV-EGFP to transfect a NPC cell line, C666-1. Then C666-1 cells were transfected by rAAV-shRNA-LMP-1 at MOI titre and the inhibiting efficiency of target gene's expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). C666-1 cells treated by RNAi on LMP-1 in vitro was directly inoculated into the liver via laparotomy under direct vision to establish the animal model of NPC xenograft in liver and lung metastasis from the liver. The biological effect and its mechanism after "gene silencing" of LMP-1 on NPC cells tumorigenesis and metastasis were observed by the primary intrahepatic tumor formation and lung metastasis and the expression of matrix metalloproteinases-9 (MMP-9) revealed by immunohistochemistry.


RESULTS
The transfection efficiency was higher than 95% with 5 x 10(4) virus genome (v.g)/cell with rAAV-EGFP. The expression of target gene was inhibited more than 90%, assessed by RT-PCR after transfection with rAAV-shRNA-LMP-1 at a dose of 5 x 10(4) v.g/cell. The primary tumor volume implanted in the liver of rAAV-shRNA-LMP-1 treatment was (0.2527 +/- 0.1152) cm3, with no significant difference in comparrison with rAAV-EGFP control group [(0.2533 +/- 0.0754) cm3, P>0.05]. But the rate of intrahepatic tumor formation was 50.0% and the rate of lung metastasis was 33.3% of the rAAV-shRNA-LMP-1 group, significantly lower than those in the rAAV-EGFP group (P<0.05). The survival time (15.50 +/- 2.47) d of rAAV-shRNA-LMP-1 group was significantly longer than that in the rAAV-EGFP group [(11.50 +/- 1.22) d, P<0.05]. Immunohistochemistry indicated suppression of LMP-1 by rAAV-shRNA-LMP-1 can down-regulate the expression of MMP-9 significantly.


CONCLUSIONS
The expression of LMP-1 can be suppressed effectively by rAAV mediated RNA interference. The suppression of LMP-1 expression has no effect on cell growth but can inhibit the metastasis in vivo, probably through down-regulating the expression of MMP-9.
Liu
Xiong
X
Department of Otolaryngology, Nanfang Hospital, Nanfang Medical University, Guangzhou 510515, China.
Li
Gang
G
Zhang
Bao
B
Wang
Lu
L
Li
Xiao-hua
XH
Li
Xiang-ping
XP
D000818:Animals; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D008175:Lung Neoplasms / Q000378:metabolism / Q000556:secondary*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009303:Nasopharyngeal Neoplasms / Q000473:pathology*; D009368:Neoplasm Transplantation; D034622:RNA Interference; D034741:RNA, Small Interfering; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D034741:RNA, Small Interfering; D014763:Viral Matrix Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020780:Matrix Metalloproteinase 9