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. 2011 Apr 10;412(2):392-400.
doi: 10.1016/j.virol.2011.01.028. Epub 2011 Feb 18.

The Epstein-Barr Virus BART microRNAs target the pro-apoptotic protein Bim

Aron R Marquitz  1 , Anuja Mathur, Cyd Stacy Nam, Nancy Raab-Traub
Affiliations

The Epstein-Barr Virus BART microRNAs target the pro-apoptotic protein Bim

Aron R Marquitz et al. Virology. .

Abstract

In Epstein-Barr Virus infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are abundantly expressed and are the template for two large clusters of miRNAs. This study indicates that both of these clusters independently can inhibit apoptosis in response to etoposide in an epithelial cell line. The Bcl-2 interacting mediator of cell death (Bim) was identified using gene expression microarrays and bioinformatic analysis indicated multiple potential binding sites for several BART miRNAs in the Bim 3'UTR. Bim protein was reduced by Cluster I and the individual expression of several miRNAs, while mRNA levels were unaffected. In reporter assays, the Bim 3' untranslated region (UTR) was inhibited by both clusters but not by any individual miRNAs. These results are consistent with the BART miRNAs downregulating Bim post-transcriptionally in part through the 3'UTR and suggest that there are miRNA recognition sites within other areas of the Bim mRNA.

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Figures

Figure 1
Figure 1. The structure of the EBV BARTs
The EBV BART miRNAs are expressed from the first two introns of the BART RNAs. In order to express these miRNAs exogenenously, the DNA sequences spanning the miRNAs was cloned as two clusters into pcDNA3 expression vectors. Cluster I contains eight miRNAs from the first intron. Cluster II contains 11 miRNAs from the second intron as well as miR-BART18 which is just upstream of exon 2. miR-BART2, which is contained in the intron between exons 4 and 5, and miR-BART21, which is upstream of miR-BART18 were not included in this study.
Figure 2
Figure 2. Generation of stable cell lines that express the BART miRNAs
AGS cells were transfected with two pcDNA3 vectors containing Cluster I or Cluster II as well as appropriate controls to generate four stable cell lines: Cluster I and Cluster II expressed together, Cluster I and II by themselves each with a control vector, and a double control vector line. A. Northern blot analysis of total RNA from the four cell lines for a miRNA representative of each cluster. RNA from the EBV infected NPC cell line, C666-1, was used as a positive control for expression. B. Quantitative RT-PCR for the miRNAs listed above each graph. C666-1 RNA was diluted with AGS RNA that lacks BART miRNAs to 50%, 20%, 10%, and 4% of the total input RNA. For each miRNA the actual cycle thresholds obtained are plotted against the theoretical cycle thresholds expected for each dilution. As a control, qRT-PCR was also performed for the small nuclear RNA U5A, which would not be expected to differ in abundance between the C666-1 and AGS cell lines. C. Quantitative RT-PCR of the AGS cell lines. RNA from the four stable cell lines was assayed for the abundance of mature miRNAs using the same qRT-PCR system. Graphed is the mean relative abundance of each miRNA in each cell line compared to control C666-1 cells for three independent measurements. Error bars represent the standard error of the mean.
Figure 3
Figure 3. Treatment of AGS cell with etoposide
The AGS stable cell lines were grown in culture in the presence of 5 or 25 μM etoposide for 24 hours. A. Control and cells expressing both clusters of miRNAs were harvested and assayed for the induction of apoptosis by assessing the amount of cleaved PARP, a caspase 3 substrate. Protein lysates were run on a SDS-PAGE gel and analyzed for cleaved PARP by Western blotting. B. Cells from all four cell lines were harvested and assessed for cleaved PARP as in A (top panel). Lysates from the experiment shown were also run on a separate gel and probed with an antibody that recognizes both the pro- and mature form of caspase-3 (bottom panel). C. PARP western blot in B was analyzed by densitometry using Image J software. The percentage of total PARP that is cleaved for each cell line and each treatment condition is presented.
Figure 4
Figure 4. Regulation of BH3-only proteins in AGS cells expressing miRNA clusters
A. Western blot analysis of AGS stable cell lines. Protein lysates were analyzed for expression of BimEL, PUMAα+β, and Bik. In each case, GRP78 was used as a loading control. B. Immunoblots for BimEL and PUMAα+β were quantitated by densitometry using Image J software. Expression levels were normalized to GRP78 loading control. Plotted is the mean of three independent experiments with error bars representing the standard error of the mean. C. Total RNA was prepared from each of the four stable AGS cell lines and qRT-PCR for Bim mRNA was performed. The mean expression level relative to the pcDNA3 control cells in three independent experiments is presented graphically. Error bars represent standard error of the mean.
Figure 5
Figure 5. Effects mediated through the Bim 3’UTR
A. A diagram of the Bim mRNA drawn to scale. The green box represents the coding region of the message. Potential miRNA binding sites predicted by PITA are indicated at their relative positions in the 3’UTR. Fragments of the message cloned into reporter vectors are indicated below. B. The indicated luciferase reporter vectors were transfected into AGS stable cells and assayed for luciferase activity after 48 hours. Each value represents the ratio of Renilla luciferase/control Firefly luciferase normalized to the control vector in each individual cell line. Plotted is the mean of at least six independent experiments. Error bars represent standard error of the mean.
Figure 6
Figure 6. Regulation of Bim in single miRNA expressing cells
A. Expression levels for the AGS stable cell lines expressing individual miRNAs by quantitative RT-PCR. RNA from each of the stable cell lines listed was assayed for the abundance of mature miRNAs using qRT-PCR. For cell lines that express multiple miRNAs, each bar represents expression of a different miRNA, in the order listed on the x axis of the graph. The relative abundance of each miRNA in each cell line compared to control C666-1 cells from three independent experiments is graphed. B. Western blot analysis of AGS cell lines expressing single miRNAs. Protein lysates were probed for expression of BimEL, PUMAα+β, and GAPDH as a loading control. C. Immunoblots for BimEL were analyzed by densitometry using Image J software. The relative signals were normalized to GAPDH loading control. Plotted is the mean of six independent experiments. Error bars represent standard error of the mean. D. Luciferase reporter vectors were transfected into AGS stable cells indicated and assayed for luciferase activity after 48 hours. Each value represents the ratio of Renilla luciferase/control Firefly luciferase normalized to the control vector in each individual cell line. Plotted is the mean of two independent experiments. Error bars represent standard error of the mean.
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