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A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
Published on: March 10, 2017
Fluorescence visualization screening for EBV-LMP1-targeted DNAzymes
Xi You1, Yu Cheng Yang, Xia Ke
1Department of Otolaryngology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Objectives:
To develop a novel screening method for DNAzymes targeting the LMP1 carboxy region.
Study Design:
To design a method to screen special DNAzymes toward the Epstein-Barr virus (EBV)-associated carcinoma before clinic use.
Setting:
Key Laboratory of the Ministry of Education-Molecular Biology of Infectious Diseases in Chongqing Medical University.
Subjects And Methods:
Four novel 10-23 DNAzymes (DZ509, DZ1037, DZ893, and DZ827) targeting the EBV-LMP1 gene were designed and evaluated by detecting enhanced green fluorescence protein (EGFP) expression of LMP1 mRNA and the protein in the nasopharyngeal carcinoma (NPC) cell line CNE2 transfected with the pEGFP-C1-LMP1c vector. The screened specific DNAzymes were then transfected into NPC cell lines C666-1 while a mutant oligonucleotide mutDZ509 and an antisense oligonucleotide ASODN509 were designed as positive and negative controls. Cell proliferation, cell apoptosis, LMP1 mRNA, and the protein were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V-fluorescence isothiocyanate (FITC), reverse transcription polymerase chain reaction (RT-PCR), and Western blots.
Results:
The inhibition rates of fluorescence expression of the DNAzymes DZ509, DZ1037, DZ893, and DZ827 were 91.25%, 65.84%, 49.02%, and 44.56%, respectively. The results were in accordance with the inhibition effects of mRNA and protein expression. The screened DZ509 could effectively knock down endogenous LMP1 expression in C666-1 cells, inhibit cell proliferation, and induce cell apoptosis compared with mutDZ509 and ASODN509.
Conclusion:
LMP1 could present a potential target for DNAzymes toward the EBV-associated carcinoma, and the EGFP expression vector could be a visible method for screening special DNAzymes before clinic use.
Insights
Researchers developed a novel screening method for DNAzymes targeting Epstein-Barr virus (EBV)-associated carcinoma. The DNAzyme DZ509 effectively inhibited LMP1 expression, proliferation, and induced apoptosis in nasopharyngeal carcinoma cells.
Area of Science:
- Molecular Biology
- Antiviral Therapy
- Cancer Research
Background:
- Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC).
- The EBV-encoded latent membrane protein 1 (LMP1) is a key oncogenic driver in EBV-associated cancers.
- Targeting LMP1 offers a potential therapeutic strategy for NPC.
Purpose of the Study:
- To develop and validate a novel screening method for DNAzymes targeting the LMP1 carboxy region.
- To identify specific DNAzymes capable of inhibiting LMP1 expression and function.
- To evaluate the potential of these DNAzymes as therapeutic agents for EBV-associated carcinoma.
Main Methods:
- Four novel 10-23 DNAzymes (DZ509, DZ1037, DZ893, DZ827) were designed to target EBV-LMP1.
- An enhanced green fluorescence protein (EGFP) expression system was used to screen DNAzyme efficacy in NPC cells.
- The most effective DNAzyme (DZ509) was further evaluated in C666-1 NPC cells for its effects on cell proliferation, apoptosis, LMP1 mRNA, and protein levels.
Main Results:
- DNAzyme DZ509 demonstrated the highest inhibition rate (91.25%) of EGFP expression, correlating with reduced LMP1 mRNA and protein levels.
- DZ509 significantly inhibited cell proliferation and induced apoptosis in C666-1 cells.
- The screened DNAzyme DZ509 showed superior efficacy compared to a mutant oligonucleotide and an antisense oligonucleotide control.
Conclusions:
- DNAzymes targeting LMP1 represent a promising therapeutic approach for EBV-associated carcinoma.
- The EGFP expression vector provides a visible and effective method for screening specific DNAzymes prior to clinical application.
- This novel screening strategy facilitates the development of targeted therapies for viral-associated cancers.
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