To determine the expression pattern of EBV miRNAs in type I and III EBV-related lymphomas, and to investigate their role in EBV pathogenesis, we initially used two BL early-passage cell lines (BL-5 and BL-8) derived from primary tumors that carry the typical t8:14 chromosomal translocation (; ref. 13
). Serially passaged primary cultures evolved from single-cell suspension (BL-5) to macroscopic clumps (BL-5R), indicating a drift to lymphoblastoid phenotype (). The expression of viral-encoding proteins, EBNA3C, EBNA2, and LMP-1, is associated with the lymphoblastoid phenotype (11
). We therefore assessed the transcription of these mRNAs in BL-5 and its lymphoblastoid derivative BL-5R by RT-PCR. Although cultured BL-5 cells contained some detectable EBNA2
transcripts, the BL-5R cells, by comparison, expressed higher levels of EBNA2
(). Consistent with its role in the lymphoblastoid phenotype, LMP-1
was exclusively expressed in BL-5R cells () and LMP-1 protein expression was confirmed by Western blot (data not shown).
Figure 1 Cytogenetic and morphologic analysis of BL lines and differential expression of EBV genes in latency type I and III BL lines. A, cytogenetic analysis of BL lines BL-5 and BL-8 reveals the characteristic t8:14 c-MYC translocation. Arrows point to translocated (more ...)
Small RNAs (<200 nucleotides) were purified from BL-5, BL-8, and BL-5R cells and EBV miRNA levels were determined by RPA. We observed elevated levels of BART2 miRNA in the parental type I BL-5 cells and a marked expression of BHRF1-3 miRNA (and concomitantly low levels of BART2 miRNA) in BL-5R cells (, lanes 2
). The early-passage type I BL-8 cells also expressed high levels of BART2 miRNA, but BHRF1-3 miRNA was not detectable in these cells (, lane 1
). This finding was in agreement with recently published data on long-term established EBV+
cell lines that showed an increased level of BHRF1 miRNAs in type III latency cell lines (8
Figure 2 Differential expression of BHRF1-3 and BART2 miRNAs in BL cell lines. A, RPA analysis of BHRF1-3 and BART2 miRNA expression in BL lines BL-8 and BL-5, a lymphoblastoid derivative BL-5R, an EBV+ DLBCL-1(+), and an EBV− DLBCL-4(−). Expression (more ...)
We also analyzed viral miRNAs from an EBV+ primary DLBCL-1 biopsy. RPA analysis revealed high levels of both BART2 and BHRF1-3 miRNAs in this tumor (, lane 4), but neither miRNA was detectable in its EBV− DLBCL counterpart (DLBCL-4; , lane 5).
To verify the differential expression of the BHRF1-3 and BART2 miRNAs by another method, we used real-time QPCR. Primers specific for the BHRF1-3, BART1, and BART2 pre-miRNAs were designed. As control for RNA isolation, we used published primers against pre-miRNAs for cellular miRNAs, miR16, miR124, and miR219 (19
). All quantitative analyses were normalized to account for differences in cell number, RNA quality, and reverse transcription efficiency (17
). We used U6 as the common denominator for these studies because it was the most abundant RNA, normally distributed and the least variable RNA in our target set (data not shown). Control experiments were done to discount mRNA or DNA contamination of miRNA QPCR amplification (data not shown). The miRNA QPCR confirmed the marked induction (~21-fold) of BHRF1-3 pre-miRNA in BL-5R cells relative to BL-5 cells. BART pre-miRNAs did not show marked differences between BL-5 and BL-5R (<2-fold) compared with BHRF1-3 pre-miRNA in the QPCR assay (). In contrast to the viral pre-miRNAs, none of the three cellular pre-miRNAs showed a pattern that correlated with EBV status, indicating that specific viral miRNA regulation rather than global defects in miRNA processing accounts for the pattern of viral miRNA levels. We also analyzed several well-characterized PEL cell lines, wherein EBV assumes a tight latency I phenotype based on latent EBV mRNA splicing patterns (18
). BART1, BART2, and, to a lesser degree, BHRF1-3 pre-miRNAs were detectable in the EBV+
PEL cell lines BC-1 and JSC-1, but not in the EBV−
BCBL-1 and BC-3 cell lines, and the expression levels were comparable with the latency I BL-5 cells (data not shown).
As all prior studies were conducted entirely in long-term culture adopted cell lines, we assessed the expression of EBV miRNAs in primary B-cell lymphomas classically associated with EBV. Endemic-type pediatric BLs from an area of high incidence and DLBCLs from HIV+ patients were confirmed to be EBV+ by in situ analysis of constitutively expressed EBV-encoded small RNAs (EBER; ) and by RT-PCR of EBV-encoded mRNAs (data not shown). As a control, an EBV (EBER)-negative DLBCL (DLBCL-4) was used in these analyses (). Each of the EBV+ DLBCLs expressed high levels of LMP-1 (), confirming that they are associated with type III EBV latency pattern. We also analyzed a primary EBV+ PEL specimen (PELmo1) as well as the established EBV+ PEL line (BC-1). RPA analysis showed that, as was observed in the EBV-associated BL lines (BL-5 and BL-8), endemic-type BLs (BL-1, BL-2, BL-3, BL-4, and BL-6) also expressed predominantly BART2 miRNA (, lanes 2, 3, 4, 5, and 7), whereas the EBV+ DLBCLs (DLBCL-1, DLBCL-2, and DLBCL-3) expressed both BART2 and BHRF1-3 miRNAs at high levels (, lanes 1, 6, 9, 11, and 12). The established PEL line and the primary PEL specimen both expressed predominantly BART2 miRNAs (, lanes 14 and 15), consistent with the reported type I EBV latency transcription pattern in PEL. In EBV− controls (DLBCL-4 and BJAB), expression of both viral miRNAs was not detected (, lanes 8, 10, and 13). Therefore, as in the type I latency cell line (e.g., BL-5), both EBV-associated primary BLs and PELs express high levels of BART2 miRNA, whereas the expression of BHRF1-3 miRNA is markedly elevated in the type III latency cell line (BL-5R) as well as in primary EBV+ AIDS-related DLBCLs. The types of primary lymphomas and cell lines analyzed and their expression pattern of EBV miRNA are summarized in .
Figure 3 Differential expression of BHRF1-3 and BART2 miRNAs in primary unmanipulated clinical specimens. A, representative immunohistochemical analysis in primary unmanipulated clinical specimens. Endemic BLs (BL-1, BL-2, and BL-3) and primary DLBCLs (DLBCL-1 (more ...)
EBV miRNA expression in primary lymphomas and cell lines
The IFN-inducible T-cell attracting chemokine CXCL-11/I-TAC is one of three CC chemokines, including CXCL-9/Mig and CXCL-10/IP-10, that bind to and activate the chemokine receptor CXCR3, which is predominantly expressed on T cells (21
). Although these cytokines are functionally redundant, CXCL-11/I-TAC is by several orders of magnitude the most potent T-cell chemo-attractant (21
). Based on published in silico
analysis, the 3′-UTR of CXCL-11/I-TAC
contains a sequence that is 100% complimentary to the BHRF1-3 miRNA sequence and therefore constitutes a putative BHRF1-3 miRNA target (7
). Because of significantly differing BHRF1-3 miRNA levels, we hypothesized that CXCL-11/I-TAC might also be differentially expressed in BL-5 and BL-5R. The basal level of CXCL-11/I-TAC proteins secreted from culture supernatants of BL-5 and BL-5R was analyzed using ELISA. BL-5 cells expressed higher levels of CXCL-11/I-TAC than BL-5R cells (), suggesting that the expression of CXCL-11/I-TAC in these cells inversely correlates with the expression of EBV-encoded BHRF1-3 miRNA.
Figure 4 Differential expression and modulation of putative BHRF1-3 miRNA target CXCL-11/I-TAC. A, ELISA analysis of basal level expression of CXCL-11/I-TAC proteins secreted from culture supernatants of BL-5 and BL-5R. B, RT-PCR analysis of CXCL-11/I-TAC mRNA (more ...)
We investigated the effect of BHRF1-3 miRNA on its putative target, CXCL-11/I-TAC. First, we studied the high BHRF1-3 miRNA BL-5R cell line. BL-5R cells were transfected with increasing amounts of BHRF1-3 miRNA antisense oligonucleotide (BHRF1-3 anti-miR) at ~65% efficiency (, top) and the CXCL-11/I-TAC level was assessed by RT-PCR. By competing the BHRF1-3 miRNA targeting sequence with its antisense RNA oligonucleotide, we repeatedly elicited basal induction of CXCL-11/I-TAC expression in BL-5R (, bottom, lanes 6–9). Control transfections with a scrambled RNA oligonucleotide (, bottom, lanes 2–5), as well as mock transfection (, bottom, lane 1), showed no modulation of CXCL-11/I-TAC expression. This result indicates that BHRF1-3 miRNA antisense competes out BHRF1-3 miRNA and reverses its repressive effect on CXCL-11/I-TAC expression. We also analyzed the expression of another chemokine, CXCL-7, in the same set of experiments. RT-PCR showed that CXCL-7 mRNA level was not modulated by transfection of either BHRF1-3 anti-miR or scrambled oligonucleotides (, bottom), which indicates that the effect of BHRF1-3 miRNA on CXCL-11/I-TAC expression is specific. Similarly, CXCL-11/I-TAC expression was markedly induced in the low BHRF1-3 miRNA BL-5 cells transfected with BHRF1-3 anti-miR, and we also observed a much greater induction of CXCL-11/I-TAC expression in BL-5 cells dually treated with both the antisense oligo of BHRF1-3 miRNA and IFN-γ (data not shown). This showed that expression of CXCL-11/I-TAC is suppressed by the EBV-encoded BHRF1-3 miRNA.
We further examined the direct effect of BHRF1-3 miRNA on CXCL-11/I-TAC expression. CXCL-11/I-TAC was stimulated to a higher level in BL-5 cells when transfected with 100 nmol/L BHRF1-3 anti-miR and induced with 1,000 units/mL IFN-γ (, left and right, lanes 2–5). Cells were then transfected with increasing amounts of BHRF1-3 miRNA sense oligos (, left and right, lanes 3–5) and analyzed for CXCL-11/I-TAC by RT-PCR (, left) and ELISA (, right). We observed a significant reduction of CXCL-11/I-TAC at both mRNA and protein levels when cells were transfected with BHRF1-3 miRNA. This result further shows that CXCL-11/I-TAC expression is suppressed by EBV miRNA BHRF1-3, suggesting that cellular chemokines can be targeted by the viral miRNA.