Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Nov;11(11):1174-84.
doi: 10.1593/neo.09888.

Modulation of LMP2A expression by a newly identified Epstein-Barr virus-encoded microRNA miR-BART22

Raymond Wai-Ming Lung  1 , Joanna Hung-Man Tong, Ying-Man Sung, Pak-Sing Leung, David Chi-Heng Ng, Shuk-Ling Chau, Anthony Wing-Hung Chan, Enders Kai-On Ng, Kwok-Wai Lo, Ka-Fai To
Affiliations

Modulation of LMP2A expression by a newly identified Epstein-Barr virus-encoded microRNA miR-BART22

Raymond Wai-Ming Lung et al. Neoplasia. 2009 Nov.

Abstract

Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1, and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remains largely unclear. The recent discovery of EBV-encoded viral microRNA (miRNA) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNA. Using large-scale cloning analysis on EBV-positive NPC cells, two novel EBV miRNA, now named miR-BART21 and miR-BART22, were identified. These two EBV-encoded miRNA are abundantly expressed in most NPC samples. We found two nucleotide variations in the primary transcript of miR-BART22, which we experimentally confirmed to augment its biogenesis in vitro and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. More importantly, we determined that the EBV latent membrane protein 2A (LMP2A) is the putative target of miR-BART22. LMP2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells; down-modulation of LMP2A expression by miR-BART22 may permit escape of EBV-infected cells from host immune surveillance. Taken together, we demonstrated that two newly identified EBV-encoded miRNA are highly expressed in NPC. Specific sequence variations on the prevalent EBV strain in our locality might contribute to the higher miR-BART22 expression level in our NPC samples. Our findings emphasize the role of miR-BART22 in modulating LMP2A expression, which may facilitate NPC carcinogenesis by evading the host immune response.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Hairpin structures and expression of novel ebv-miRNA. (A) Stem-loop structures of miR-BART21 and miR-BART22 are illustrated. The cloned mature miRNA sequences are indicated by underlining. The folding energy (dG) and EBV genomic sequences are listed below the figures. (B) Representative Northern blot for cell lines, NPC xenografts and NPC biopsies showing the expression of miR-BART21 and miR-BART22 are displayed. The normal epithelial cell line NP460 was included as a negative control. U6 RNA or SYBR Gold staining PAGE was used to control for RNA loading. The miRNA expression in the same sample was assayed by QRT-PCR and was compared with C666-1 (set at 100). The analysis of each sample was performed in triplicate with the mean ± SD shown.
Figure 2
Figure 2
miR-BART10 and miR-BART22 expression in C666-1 and Namalwa cells. (A) Northern blot analysis demonstrates that the expression of miR-BART22 is significantly higher than miR-BART10 in C666-1 (P < .05) but not in Namalwa cells. Representative results from three independent experiments are shown. The relative expression level for each miRNA is calculated with reference to Namalwa (set as 1) after normalization with U6 level. (B) Expression ratio of miR-BART22 to miR-BART10 is significantly higher in C666-1 than in Namalwa cells as measured by QRT-PCR (P < .05). The expression of miR-BART22 is compared with miR-BART10 after normalization with EBNA1. The results shown are the mean ± SD from three independent experiments.
Figure 3
Figure 3
The nucleotide polymorphisms in pri-miR-BART-21 and 22 from C666-1-EBV. (A) The nucleotide variations in pri-miR-BART21 (AJ507799, 145435:145614; left) and pri-miR-BART22 (147126:147305; right) are shown in shaded color. The mature miRNA sequences are shown in gray boxes. (B) The predicted secondary structures of C666-1-EBV miR-BART22 (EU828629, 8087:8267; left), Raji-EBV miR-BART22 (AJ507799, 147137:147456; middle), and the two nucleotide variations of Raji-EBV, Raji-EBV-2nt mut (147144 A > T and 147146 C > A; right) are shown. The folding energy (dG) with units (kcal/mol) is indicated. (C) In vitro Drosha/DGCR8 processing of the pri-miR-BART22 transcript. C666-1-EBV and Namalwa-EBV pri-miR-BART22 transcripts (pri-RNA) (AJ507799, 147137:147456) were in vitro-transcribed and incubated with bead-bound Flag-tagged Drosha/DGCR8 complex (+) or beads containing empty vector transfected cell extract (-). The digested products were separated on 8% PAGE for analysis (left). The presence of pre-miR-BART22 with a size around 70 nt was confirmed by Northern blot with a miR-BART22 complementary oligonucleotide probe (right). The representative result from at least three individual blots is shown in panel C.
Figure 4
Figure 4
Viral LMP2A is a potential target of miR-BART22. (A) Base complementarity suggests that the putative miR-BART22 target site is in the 3′UTR of LMP2A (AJ507799, 5545:5568; upper). The wild-type (LMP2A-WT), truncated (LMP2A-M1) and mutated (LMP2A-M2 and -M3) LMP2A sequences are shown. The seed-binding region is underlined and the base substitutions a remarked in red (lower). (B) The sequences of miRNA mimics are displayed. The base substitutions made to restore seed mutant complementarity are labeled in red (miR-M2-BART22 and miR-M3-BART22). The relative firefly luciferase activity was normalized to the renilla luciferase control, and results were taken from at least three independent experiments. Data shown are the mean ± SD. Statistical analysis by Student's t test was used. **P < .001.
Figure 5
Figure 5
LMP2A protein expression in NPC, IM, and HD specimens. Upper panel: EBV-positive NPC cell line and xenografts. LMP2A expression was not detected in NPC cell line C666-1 and xenograft X666. Only weak and focally LMP2A expression was detected in xenograft X2117. Middle panel: Primary NPC biopsies. Weak expression of LMP2A was noted in three NPC samples (NPC6, 8, 11). A negative example (NPC2) was included. Lower panel: IM and HD. Strong LMP2A expression was detected in IM. Three positive examples of HD were included.
Figure 6
Figure 6
Suppression of LMP2A protein expression by miR-BART22. (A) Western blot of LMP2A and EGFP in 293FT cells transiently cotransfected with 0.1 µg per well pEGFP-C1 plasmid in six-well plate format, the amount of expression plasmids as indicated (lanes 3–6), and 2 µg of total DNA made up with pcDNA3.1 as carrier. LMP2A expression level was normalized to EGFP. miR-BART22 and LMP2A RNA expression levels in the same sample were analyzed by QRTPCR and are shown relative to lane 3. (B) The representative Western blot results indicate that miR-BART22 represses LMP2A expression in HEK293-LMP2A stable clones. The stable clones transfected with the siRNA control (siCtl) and LMP2A specified siRNA (siLMP2A) were included as controls. The LMP2A expression level was normalized to actin and the level relative to mock transfection (set at 1) was calculated. LMP2A mRNA expression in the same sample was assayed by QRT-PCR.
Figure 7
Figure 7
Function of ebv-BART-miRNA on host cell survival. miR-BART1, 16, and 17 can regulate the viral LMP1 protein expression [12] and miR-BART5 can alter the expression of the p53-upregulated modulator of apoptosis (PUMA) [13]. And in this article, miR-BART22 is demonstrated as a modulator of the viral gene, LMP2A.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. - PubMed
    1. Lo KW, To KF, Huang DP. Focus on nasopharyngeal carcinoma. Cancer Cell. 2004;5:423–428. - PubMed
    1. Raab-Traub N. Epstein-Barr virus in the pathogenesis of NPC. Semin Cancer Biol. 2002;12:431–441. - PubMed
    1. Karran L, Gao Y, Smith PR, Griffin BE. Expression of a family of complementary-strand transcripts in Epstein-Barr virus-infected cells. Proc Natl Acad Sci USA. 1992;89:8058–8062. - PMC - PubMed
    1. Hitt MM, Allday MJ, Hara T, Karran L, Jones MD, Busson P, Tursz T, Ernberg I, Griffin BE. EBV gene expression in an NPC-related tumour. EMBO J. 1989;8:2639–2651. - PMC - PubMed

Publication types

Substances

LinkOut - more resources