Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt's lymphoma cells

Abstract

Purpose: Epstein-Barr virus (EBV) is associated with the development of several lymphoid and epithelial malignancies, including Burkitt's lymphoma. The EBV latent protein, EBV Nuclear Antigen 1 (EBNA1), is detectable in almost all types of EBV-associated tumors and is essential for replication and maintenance of the latent episome of EBV. We here examined whether the RNA interference (RNAi) technique could be employed to suppress expression of EBNA1 in EBV-positive Burkitt's lymphoma cells.

Methods: A Raji cell line expressing small hairpin RNAs (shRNAs) against EBNA1 was established and EBNA1 mRNA level was determined by real-time RT-PCR analysis. We investigated the effects of EBNA1 silence on lymphoma cell growth and cell cycle progression.

Results: Transfection of an EBNA1 RNAi plasmid resulted in substantial loss of EBNA1 mRNA and significantly inhibited proliferation of Raji cells relative to the control plasmid case. Suppression of EBNA1 was also associated with downregulation of EBV oncogene EBNA2, a decreased PCNA labeling index and increased G0/G1 fraction in cell cycle analysis.

Conclusions: These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.

Fig. 1

Designs of the EBNA1 shRNA template and real-time PCR Primers. a The predicted secondary structure of the shRNA against EBNA1. Since hairpin sequences are difficult to sequence, three nucleotides in the target sense sequence were substituted to facilitate insert verification. b Model for transcription and splicing of EBNA1 mRNAs. The top bar illustrates the linear form of the EBV genome and the positions of four different promoters (bent arrows). The lower panel shows three classes of EBNA1 transcripts and their corresponding infection types. Coding regions are indicated by boxes and excised intron sequences by thin lines. c Partial cDNA sequence of the 5′ end of the EBNA1 mRNA (GenBank accession number M13941). The sequences of the PCR primers and TaqMan probe used for EBNA1 quantitation are underlined. The splice site between exons U and K is indicated with an arrowhead. The start codon of EBNA1 ORF is shown translated. d Specificity and efficiency of PCR primers designed for real-time PCR analysis. GAPDH is amplified as an internal standard. Negative water blanks were included in each analysis

Fig. 2

Suppression of EBNA1 expression in Raji cells by RNA interference. a Quantitation of EBNA1 mRNA by real-time RT-PCR analysis. Values represent the mean ± standard deviation (SD) of two separate experiments performed in duplicate. **P<0.01 versus the control cells. Significance was determined using the Student’s t-test. b Cells stably transfected with EBNA1 RNAi or control vector were extracted and analyzed by Western blotting for EBNA1 and actin as a loading control. c Relative amounts of EBNA1 protein in Fig. 2b as determined by densitometry. Data are mean ± SD from three separate experiments. **P<0.01 versus the control cells. Significance was determined by using Student’s t-test

Fig. 3

Effects of EBNA1 RNAi on proliferation of Raji cells. a Cell growth curve assays. EBNA1-targeted RNA interference significantly inhibited the proliferation of Raji cells over four days (time×treatment interaction: P=0.0001). Data are mean ± SD from triplicate assays and are representative of two independent experiments. Significance was determined using repeated ANOVA (analysis of variance). b Downregulation of EBNA2 protein. Cells stably transfected with EBNA1 RNAi or control vector were extracted and analyzed by Western blotting for EBNA2 and α-Tubulin as a loading control. c Ki-67 immunoreactivity and PCNA labeling index determined by immunocytochemistry. Raji cells in three random fields of each slide were counted and the percentage of PCNA-positive cells was calculated and expressed as the mean ± SD. ***P<0.001. Significance was determined using the Student’s t-test

Fig. 4

Suppression of EBNA1 expression in Raji cells results in increased frequency of G0–G1 phase cells. FACS analysis of the cell cycle distribution using a BrdU Flow Kit. Data are mean ± SD from three separate experiments. **P<0.01 versus the control cells. Significance was determined using the Student’s t-test