The Epstein Barr-encoded BART-6-3p microRNA affects regulation of cell growth and immuno response in Burkitt lymphoma

Abstract

Background: Burkitt lymphoma is an aggressive B-cell lymphoma presenting in three clinical forms: endemic, sporadic and immunodeficiency-associated. More than 90% of endemic Burkitt lymphoma carry latent Epstein-Barr virus, whereas only 20% of sporadic Burkitt lymphoma are associated with Epstein-Barr infection. Although the Epstein-Barr virus is highly related with the endemic form, how and whether the virus participates in its pathogenesis remains to be fully elucidated. In particular, the virus may impair cellular gene expression by its own encoded microRNAs.

Methods: Using microRNA profiling we compared Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for both cellular and viral microRNAs. The array results were validated by qRT-PCR, and potential targets of viral microRNAs were then searched by bioinformatic predictions, and classified in functional categories, according to the Gene Ontology. Our findings were validated by in vitro functional studies and by immunohistochemistry on a larger series of cases.

Results: We showed that a few cellular microRNAs are differentially expressed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases, and identified a subset of viral microRNAs expressed in Epstein-Barr-positive Burkitt lymphomas. Of these, we characterized the effects of viral BART6-3p on regulation of cellular genes. In particular, we analyzed the IL-6 receptor genes (IL-6Rα and IL-6ST), PTEN and WT1 expression for their possible relevance to Burkitt lymphoma. By means of immunohistochemistry, we observed a down-regulation of the IL-6 receptor and PTEN specifically in Epstein-Barr-positive Burkitt lymphoma cases, which may result in the impairment of key cellular pathways and may contribute to malignant transformation. On the contrary, no differences were observed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for WT1 expression.

Conclusions: Our preliminary results point at an active role for the Epstein-Barr virus in Burkitt lymphomagenesis and suggest new possible mechanisms used by the virus in determining dysregulation of the host cell physiology.

Keywords: Burkitt lymphoma; EBV; MicroRNAs.

Figure 1

microRNA profiling of EBV-positive vs. EBV-negative BL cases. (a) Heatmap of miRNA differential expression in EBV-positive BL versus EBV-negative BL cases. 12 EBV-negative and 6 EBV-positive BL cases were compared in terms of viral and human miRNA expression using the Agilent® microRNA expression microarray technology. While each column represents a BL case, rows represent human miRNAs differentially expressed in two groups or viral miRNAs expressed in EBV-positive cases. We recognized 8 out of 470 cellular miRNA deregulated in EBV-positive and -negative BLs (FDR < 0.05). Indeed 13 out of 46 EBV miRNAs were found to be expressed in EBV-positive BL cases. (b) Validation by RT-qPCR of the array results on the cellular miRNAs differentially expressed between EBV-positive and EBV-negative BL cases. The graph is representative of three different qRT-PCR experiments. Error bars represent standard deviation between duplicates.

Figure 2

Immunohistochemistry of BART6-3p target genes. The expression level of p80, gp130 and PTEN was evaluated in EBV-positive (right) and EBV-negative (left) BL cases. p80 was strongly expressed in the cytoplasm (upper panel), gp130 showed membrane and cytoplasmic positivity (middle panel), PTEN was found in the nucleus (lower panel). Macrophages were used as internal positive controls. p80, gp130 and PTEN were found to be meaningfully down-regulated in EBV positive BL cases (right panel, upper, middle and lower images, respectively). (400x magnification).

Figure 3

BART6-3p-regulated pathways in BL-derived cell lines. (a) The expression of BART6-3p was evaluated in two EBV-negative BL-derived cell lines (Akata 2A8 and Ramos) and two EBV-positive BL-derived cell lines (Akata and Raji), to select that expressing BART6-3p at the highest level. The Akata cell line shows higher expression level of BART6-3p. (b): qRT-PCR of BART6-3p target genes following BART6-3p inhibition. A BART6-3p antagomir was transfected in Akata cells and the expression level of p80, gp130 and PTEN was evaluated 24 hrs post-transfection. Cells transfected with BART6-3p inhibitor showed a marked up-regulation of p80, gp130 and PTEN, in respect with cells transfected with the negative control (NC), confirming regulation of these genes by BART6-3p. As a control, down-regulation of the endogenous BART6-3p was observed following transfection with the antagomir. The graph is representative of three different qRT-PCR experiments. Error bars represent standard deviation between duplicates. (c): Inhibition of BART6-3p by its antagomir results in the activation of the downstream pathways of the IL-6 receptor (p80 and gp130) and PTEN. The expression of p80, gp130 and PTEN was evaluated at the protein level by WB. In addition, two downstream signaling pathways were also evaluated following BART6-3p inhibition, the activation of NF-κB, which occurs through IκBα phosphorylation, and the decrease of the phosphorylated form of Akt, which acts downstream from PTEN. Protein levels were evaluated 48 hours post-transfection by Western blotting. The bands were then analyzed by the Image J software and relative expression levels were normalized to those of β-actin. Activation of NF-κB is consequent to IκB-α phosphorylation; PTEN activity results in the negative regulation of Akt, documented by a lower phosphorylation level. EBNA1 expression was measured as a control for EBV-positivity. The figure is representative of three different experiments.

Figure 4

Effects of BART6-3p on cell viability. (a) The effects of BART6-3p on cell proliferation and cell death were monitored following its inhibition by a synthetic antagomir, by Tripan blue exclusion assay. Proliferation curves show that inhibition of BART6-3p results in a decreased proliferation rate, whereas Tripan blue staining revealed an increased in the dead cell fraction (data not shown). (b) To detect whether cell death could be dependent on apoptosis, an Annexin V staining was performed 24 hrs after inhibitor transfection. Our results indicate an increase of the apoptotic fraction of cells transfected with BART6-3p inhibitor, in respect with its negative control (NC). The figure is representative of three different experiments.