miR-18a reactivates the Epstein-Barr virus through defective DNA damage response and promotes genomic instability in EBV-associated lymphomas

Abstract

Background: The Epstein-Barr virus (EBV) is closely associated with several types of malignancies. EBV is normally present in the latent state in the peripheral blood B cell compartment. The EBV latent-to-lytic switch is required for virus spread and virus-induced carinogenesis. Immunosuppression or DNA damage can induce the reactivation of EBV replication. EBV alone is rarely sufficient to cause cancer. In this study, we investigated the roles of host microRNAs and environmental factors, such as DNA-damage agents, in EBV reactivation and its association with lymphomagenesis.

Methods: We first analyzed the publicly available microRNA array data containing 45 diffuse large B-cell lymphoma patients and 10 control lymph nodes or B cells with or without EBV infection. In situ hybridization for miR-18a and immunohistochemitry were performed to evaluate the correlation between the expression of miR-18a and nuclear EBV protein EBNA1 in lymphoid neoplasm. The proliferative effects of miR-18a were investigated in EBV-positive or -negative lymphoid neoplasm cell lines. EBV viral load was measured by a quantitative real-time EBV PCR and FISH assay. The genomic instability was evaluated by CGH-array.

Results: In this study, we analyzed the publicly available microRNA array data and observed that the expression of the miR-17-92 cluster was associated with EBV status. In situ hybridization for miR-18a, which is a member of the miR-17-92 cluster, showed a significant upregulation in lymphoma samples. miR-18a, which shares the homolog sequence with EBV-encoded BART-5, promoted the proliferation of lymphoma cells in an EBV status-dependent manner. The DNA-damaging agent UV or hypoxia stress induced EBV activation, and miR-18a contributed to DNA damaging-induced EBV reactivation. In contrast to the promoting effect of ATM on the lytic EBV reactivation in normoxia, ATM inhibited lytic EBV gene expression and decreased the EBV viral load in the prescence of hypoxia-induced DNA damage. miR-18a reactivated EBV through inhibiting the ATM-mediated DNA damage response (DDR) and caused genomic instability.

Conclusions: Taken together, these results indicate that DNA-damaging agents and host microRNAs play roles in EBV reactivation. Our study supported the interplay between host cell DDR, environmental genotoxic stress and EBV.

Keywords: DNA damage response; EBV reactivation; Genomic instability; miR-18a.

Fig. 1

Expression of miR-18a in lymphoma patients. a Relative expression of miR-18a in diffuse large B-cell lymphoma patients and normal controls; publicly available microRNA array data (GSE42906) were compared between groups with GEO2R. b Unsupervised hierarchical clustering of microRNA expression. The miR-17-92 cluster and EBV-encoded microRNAs were differentially expressed between EBV- positive and -negative B cells; High and low expression levels are indicated by red and green, respectively. The raw data are shown in NCBI, GEO:GSE36926. c Expression of miR-18a and EBNA1. The expression of miR-18a was measured by in situ hybridization. The expression of EBNA1 was measured by immunohistochemistry. Representative figures are shown (× 100); Upper left and upper right: lymphoma biopsies; lower left and lower right: normal control lymph nodes. d Scatter plot of the observed expression scores of miR-18a. Expression was scored semi-quantitatively by multiplying the intensity and area of staining. e Correlation of the expression of EBNA1 and miR-18a. f Kaplan-Meier curves for patients according to the tumor expression of miR-18a. g Kaplan-Meier curves for patients according to the tumor expression of EBNA1

Fig. 2

miR-18a promotes cell proliferation in EBV-positive lymphoma cells. a miR-18a promoted tumor cell growth in vitro in EBV-positive lymphoma cell lines. CCK-8 cell viability assay was performed after the transfection of miR-18a mimics or miR-18a inhibitor into EBV-positive lymphoma cell lines (P3HR-1, Raji, EBV infected BJAB) and the EBV-negative lymphoma cell line BJAB. The data represented the mean values of five repeats. The data are shown as the means±SD (Student T-test, *p < 0.05). b Flow cytometry analysis of the cell cycle after the transfection of miR-18a. c Cell cycle distribution of cells in S phase. The data were presented as the means±SD of four replicates (Student T-test, *p < 0.05; **p < 0.01)

Fig. 3

miR-18a increases the EBV viral load. a Transfection of miR-18a mimics in P3HR-1 and Raji resulted in a higher EBV viral load. Quantitative real-time EBV PCR was performed in samples collected from the culture media of cells. Viral DNA was extracted, and PCR was carried out according to the instructions. The EBV copy number was calculated according to a standard curve. b Expression of EBNA1 after transfection of miR-18a mimics and inhibitor in Raji cells as measured by western blotting. c Relative expression of EBV gene expression after transfection of miR-18a. d Visualization of episomal and integrated EBV DNA by fluorescence in situ hybridization

Fig. 4

DNA damage reactivates EBV. a Expression of γ-H2AX as measured by immunofluorescence after treatment with hypoxia or UV. EBV-positive Raji cells were stained with anti-γ-H2AX and DAPI. Expression of γ-H2AX is indicated as green loci. DAPI was used to stain the cell nuclei. The merge images present the DAPI and FITC as blue and green, respectively. b Expression of γ-H2AX as measured by western blotting after treatment with hypoxia or UV in Raji cells. β-actin served as an loading control. c EBV copy number as measured by real-time PCR after treatment with hypoxia or UV. d Relative expression of EBV genes as measured by real-time PCR after treatment with hypoxia or UV

Fig. 5

DNA-damaging agents reactivate EBV through miR-18a. a Relative expression of miR-18a after treatment with hypoxia or UV. Real-time PCR was used to measure the mRNA expression of EBV-related genes. β-actin served as an internal control. b miR-18a inhibitor decreased the EBV copy number induced by hypoxia or UV in Raji cells. c miR-18a inhibitor decreased EBV gene expression induced by hypoxia or UV. Relative expression of EBV genes as measured by real-time PCR. upper: hypoxia treatment; lower: UV treatment

Fig. 6

miR-18a reactivates EBV by targeting ATM. a DNA-PK inhibitor AZD 8055 increased EBV gene expression in Raji cells. Real-time PCR was used to measure the mRNA expression of EBV-related genes. GAPDH served as an internal control. b ATM is a potential target of miR-18a. I: Schematic representation of the 3’UTR of ATM. The red bar shows the predicted miR-18a binding sites in the 3’UTR of ATM. The sequence of mature miR-18a aligned to target sites is shown. (II) Luciferase activity assay. The reporter constructs in which the 3’UTR of ATM, wild-type or miR-18a binding-site mutant, was cloned downstream of the luciferase open reading frame. 293 cells were co-transfected with the luciferase construct and miR-18a mimics or control miRNA. The renilla construct was also cotransfected as an internal control. Luciferase activity was normalized to renilla luciferase activity. The data were presented as the means±SD of two experiments with six replicates (Student T-test, *p < 0.05; **p < 0.01; ***p < 0.001). III: miR-18a decreased the expression of ATM. Western blot of ATM was performed 48 h after the transfection of miR-18a mimics and miR-18a inhibitor. GAPDH was used as an internal control. c ATM inhibited the expression of EBV-related genes under hypoxic conditions (*p < 0.05; **p < 0.01). d ATM reversed the promotion effect of miR-18a on the EBV-related gene expression (*p < 0.05; **p < 0.01). e EBV gene expression after transfection of ATM under normoxia (*p < 0.05; **p < 0.01). f Transfection with ATM inhibited the promotion effect of miR-18a on the EBV viral load

Fig. 7

miR-18a induces DNA damage. a Expression of γ-H2AX as measured by immunofluorescence in Raji cells; EBV-positive Raji cells were stained with anti-γ-H2AX and DAPI. Expression of γ-H2AX is indicated as green loci. DAPI was used to stain the cell nuclei. The merge images present the DAPI and FITC as blue and green, respectively. b Expression of γ-H2AX as measured by western blotting in EBV-positive or -negative cells. c Detection of DNA damage after transfection of miR-18a. The comet assay was applied. Cells were electrophoresed in agarose gels on a coverslip and were stained with propidium iodide. Labeled DNA was visualized under a fluorescence microscope. d Detection of DNA damage after transfection of miR-18a in EBV-negative and -positive BJAB cells. Magnification, × 100. e Graphic presentation of all chromosomal changes. Cells transfected with miR-18a and mimics negative control were analyzed by comparative genomic hybridization array (Array-CGH). The regions of DNA gain (blue) and loss (red) are shown