Effect of EBV LMP1 targeted DNAzymes on cell proliferation and apoptosis.
The latent membrane protein (LMP1) encoded by Epstein-Barr virus (EBV) has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. RNA-cleaving DNA enzymes are catalytic nucleic acids that bind and cleave a target RNA in a highly sequence-specific manner. In this study, we explore the potential of using DNAzymes as a therapeutic approach to EBV-associated carcinomas by targeting the LMP1 gene. In all, 13 different phosphorothioate-modified "10-23" deoxyribozymes (DNAzymes) were designed and synthesized against the LMP1 mRNA and transfected into B95-8 cells, which constitutively express the LMP1. Fluorescence microscopy was used to examine the cellular uptake and distribution in B95-8 cells. As demonstrated in Western blots, three out of 13 deoxyribozymes significantly downregulated the expression of LMP1 in B95-8 cells. These DNAzymes were shown to markedly inhibit B95-8 cell growth compared with a disabled DNAzyme and untreated controls, as determined by an alamarBlue Assay. It was further demonstrated that these DNAzymes arrested the B95-8 cells in G0/G1 using flow cytometry. Interestingly, the active DNAzymes could also downregulate the expression of Bcl-2 gene in treated cells, suggesting a close association between the LMP1 and Bcl-2 genes and their involvement in apoptosis. This was further confirmed with the result that the DNAzymes could induce the release of cytochrome c from mitochondria, which is the hallmark of the apoptosis. The present results suggest that the LMP1 may present a potential target for DNAzymes towards the EBV-associated carcinoma through cell proliferation and apoptosis pathways.
Lu
Zhong-Xin
ZX
Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, China.
Ye
Mao
M
Yan
Guang-Rong
GR
Li
Qun
Q
Tang
Min
M
Lee
Leo M
LM
Sun
Lun-Quan
LQ
Cao
Ya
Y
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D045304:Cytochromes c / Q000378:metabolism; D021881:DNA, Catalytic / Q000494:pharmacology*; D004277:DNA, Single-Stranded / Q000494:pharmacology*; D004854:Herpesvirus 4, Human / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008928:Mitochondria; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C402582:RNA-cleaving DNA 10-23; D014763:Viral Matrix Proteins; D045304:Cytochromes c