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[Inhibiting target gene expression and controlling growth of Epstein-Barr virus transformed cells by antisense RNA
Jian-jing Chen1, Nancy Raab-Traub, Qing-yun Yao
1Cancer Center, Zhongshan University, Guangzhou 510060, P. R. China. c2jj@263.net
Background & Objectives:
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.
Conclusion:
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.
Insights
Antisense RNA effectively down-regulates Epstein-Barr virus latent membrane protein (LMP) in EBV-transformed cells. This strategy inhibits LMP overexpression and cell growth, offering a potential approach for latent EBV infections and nasopharyngeal carcinoma prevention.
Area of Science:
- Molecular Biology
- Virology
- Cancer Research
Context:
- Epstein-Barr virus (EBV) latent membrane protein (LMP) is implicated in nasopharyngeal carcinoma (NPC) development.
- Investigating methods to control LMP expression is crucial for understanding EBV-associated oncogenesis.
Purpose:
- To investigate the efficacy of antisense RNA (AsRNA) in down-regulating LMP expression in EBV-transformed lymphoid cells.
- To establish an antisense system for inhibiting LMP expression and its effects on cell transformation.
Summary:
- A single-strand antisense transcription system generated abundant AsRNA.
- An in situ antisense tracing system (ATSIS) visualized AsRNA/sense RNA duplex dimers in living cells.
- AsLMPmRNA suppressed LMP overexpression, inhibited cell agglutination, and reduced MTT formation in EBV-transformed cell lines.
Impact:
- Demonstrates the effectiveness of AsLMPmRNA in down-regulating EBV-LMP and inhibiting transformed cell growth.
- Provides a potential therapeutic strategy for latent EBV infections and prevention of NPC.
- Highlights the utility of ATSIS for visualizing and validating antisense RNA activity in situ.