[Inhibiting target gene expression and controlling growth of Epstein-Barr virus transformed cells by antisense RNA transcripts].
OBJECTIVE
The latent membrane protein gene (LMP) of Epstein-Barr virus (EBV) was thought to play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). In this study, the authors investigated the effects of antisense RNA (AsRNA) on LMP for down regulating at the target gene over expression in EBV transformed lymphoid cells, and set up an antisense system to inhibit LMP expression.


METHODS
Constructing the single strand antisense transcription system in vitro, the authors have got large amount of AsRNA. Designing and setting up an antisense tracing system in situ (ATSIS), the authors could observe the living particles of AsRNA/sense RNA duplex dimer. With time lapse phase-contrast microscopy, the agglutination phenotype on living cells was easily detected by MTT test, the inhibition rate on EBV transformed cells was calculated.


RESULTS
LMP 1.9 fragment ligated into pGEM vector in reverse orientation have been constructed and produced a plentiful amount of AsLMPmRNA which could incorporated into both B95-8 and C1936 cell lines by endophagocytosis and formed the duplex dimer of As/Sense RNA. This particles have been visualized in situ when labelling 35S isotope by ATSIS. When AsLMPmRNA acted as agents for specific inhibition to LMP over expression, the transform phenotype of cell agglutination have been suppressed and MTT particle formatin was apparently reduced both two EBV tansformed cell lines.


CONCLUSIONS
AsLMPmRNA targets at sense strand have a high effectiveness of down-regulation on EBV-LMP overexpression. This down regulating function of LMP and growth inhibition on transformed cell is demonstrated by the antisenes tracing system in situ (ATSIS). The results provide a clue to overcome the latent EBV infection in human bodies all living long time and to prevent it inducing NPC in high incidence area by antisense strategies.
Chen
Jian-jing
JJ
Cancer Center, Zhongshan University, Guangzhou 510060, P. R. China. c2jj@263.net
Raab-Traub
Nancy
N
Yao
Qing-yun
QY
Zhang
Feng
F
Huang
Mei-ling
ML
Kuang
Zhu-ji
ZJ
Zhang
Xiao-shi
XS
Ye
Yan-li
YL
Gu
Li
L
D000371:Agglutination; D002455:Cell Division / Q000187:drug effects; D002472:Cell Transformation, Viral; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D009303:Nasopharyngeal Neoplasms / Q000628:therapy / Q000821:virology; D016372:RNA, Antisense / Q000494:pharmacology* / Q000627:therapeutic use; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D016372:RNA, Antisense; D014763:Viral Matrix Proteins