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.
Keywords: Epstein-Barr virus; deoxyribozyme; enhanced green fluorescent protein; gene therapy; latent membrane protein 1; nasopharyngeal carcinoma.