Cancer Res Treat.  2020 Apr;52(2):492-504. 10.4143/crt.2019.457.

EBV-miR-BHRF1-1 Targets p53 Gene: Potential Role in Epstein-BarrVirus Associated Chronic Lymphocytic Leukemia

• Xu DM
• Kong YL
• Wang L
• Zhu HY
• Wu JZ
• Xia Y
• Li Y
• Qin SC
• Fan L
• Li JY
• Liang JH
• Xu W

• KMID: 2500334
• DOI: http://doi.org/10.4143/crt.2019.457

Abstract

Purpose
The purpose of this study was to investigate the prognostic impact of Epstein-Barr virus (EBV)–microRNA (miRNA, miR)-BHRF1-1 with chronic lymphocytic leukemia (CLL) as well as role of EBV-miR-BHRF1-1 in p53 gene.
Materials and Methods
Quantitative reverse transcription–polymerase chain reaction and western blotting were used to quantify EBV-miR-BHRF1-1 and p53 expression in cultured CLL.
Results
p53 aberration was associated with the higher expression level of EBV-miR-BHRF1-1 (p < 0.001) which was also an independent prognostic marker for overall survival (p=0.028; hazard ratio, 5.335; 95% confidence interval, 1.193 to 23.846) in 97 newly-diagnosed CLL patients after adjusted with International Prognostic Index for patients with CLL. We identified EBV-miR-BHRF1-1 as a viral miRNA regulator of p53. EBV-miR-BHRF1-1 repressed luciferase reporter activity by specific interaction with the seed region within the p53 3- untranslated region. Discordance of p53 messenger RNA and protein expression was associated with high EBV-miR-BHRF1-1 levels in CLL patients and cell lines. EBV-miR-BHRF1- 1 inhibition upregulated p53 protein expression, induced cell cycle arrest and apoptosis and decreased cell proliferation in cell lines. EBV-miR-BHRF1-1 mimics downregulated p53 protein expression, decreased cell cycle arrest and apoptosis, and induced cell proliferation in cell lines.
Conclusion
This study supported the role of EBV-miR-BHRF1-1 in p53 regulation in vitro. Our results support the potential of EBV-miR-BHRF1-1 as a therapeutic target in EBV-associated CLL with p53 gene aberration.

Keyword

Chronic lymphocytic leukemia; EBV-miR-BHRF1-1; p53

Figure

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  Fig. 1. Time-to-treatment (TTT) (A) and overall survival (OS) (B) curve of 97 patients with chronic lymphocytic leukemia based on Epstein-Barr virus (EBV)–microRNA (miR)–BHRF1-1 by Kaplan-Meier estimation. Low group value is below the cut-off value (0.0012), and the high group above the cut-off value

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  Fig. 2. Epstein-Barr virus (EBV)–microRNA (miR)–BHRF1-1 targets the p53 3′-untranslated regions (3′-UTR). (A) Schematic representation of EBV-miR-BHRF1-1 putative blinding site on the 3′-UTR of p53. (B) Dual-luciferase assay performed in 293T cells suggested that EBV-miR-BHRF1-1 mimics significantly suppress the activity of p53 when compared to EBV-miRBHRF1-1-NC (negative control).

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  Fig. 3. Epstein-Barr virus (EBV)–microRNA (miR)–BHRF1-1 and p53 expressions in five chronic lymphocytic leukemia (CLL) patients. (A) Quantitative reverse transcription–polymerase chain reaction analysis of EBV-miR-BHRF1-1 expression in five CLL patients. (B) Western blot analysis of LMP1, p53 and p21, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control.

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  Fig. 4. Epstein-Barr virus (EBV)–microRNA (miR)–BHRF1-1 inhibitor up-regulate p53 expression and induced chronic lymphocytic leukemia cells apoptosis in MEC1 and JVM3 cells. Quantitative reverse transcription–polymerase chain reaction analysis of EBV-miR-BHRF1-1 (A) and p53 mRNA (B) expression in MEC1 and JVM3 cells after transfected with EBV-miR-BHRF1-1 inhibitor. (C) Western blot analysis of LMP1, p53, p-p53 and p21, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control. The distribution of cell cycle in MEC1 (D) and JVM3 (E) cells; Cell Counting Kit-8 experiments detected the proliferation ability of MEC1 (F) and JVM3 (G) cells. Apoptosis assay of MEC1 (H) and JVM3 (I) cells. Experiments were done in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 5. Epstein-Barr virus (EBV)–microRNA (miR)–BHRF1-1 mimic down-regulate p53 expression and increased S phase in MEC1 and JVM3 cells. Quantitative reverse transcription–polymerase chain reaction analysis of EBV-miR-BHRF1-1 (A) and p53 mRNA (B) expression in MEC1 and JVM3 cells after transfected with EBV-miR-BHRF1-1 mimics. (C) Western blot analysis of LMP1, p53 and p21, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control. The distribution of cell cycle in JVM3 (D) and MEC1 (E) cells; Cell Counting Kit-8 experiments detected the proliferation ability of MEC1 (F) and JVM3 (G) cells. Apoptosis assay of MEC1 (H) and JVM3 (I) cells. Experiments were done in triplicate. **p < 0.01, ***p < 0.001.

Reference

References

1. Pfeffer S, Zavolan M, Grasser FA, Chien M, Russo JJ, Ju J, et al. Identification of virus-encoded microRNAs. Science. 2004; 304:734–6.

2. Qiu J, Cosmopoulos K, Pegtel M, Hopmans E, Murray P, Middeldorp J, et al. A novel persistence associated EBV miRNA expression profile is disrupted in neoplasia. PLoS Pathog. 2011; 7:e1002193.

3. Imig J, Motsch N, Zhu JY, Barth S, Okoniewski M, Reineke T, et al. microRNA profiling in Epstein-Barr virus-associated B-cell lymphoma. Nucleic Acids Res. 2011; 39:1880–93.

4. Barth S, Meister G, Grasser FA. EBV-encoded miRNAs. Biochim Biophys Acta. 2011; 1809:631–40.

5. Navari M, Etebari M, Ibrahimi M, Leoncini L, Piccaluga PP. Pathobiologic roles of Epstein-Barr virus-encoded microRNAs in human lymphomas. Int J Mol Sci. 2018; 19:E1168.

6. Liang JH, Gao R, Xia Y, Gale RP, Chen RZ, Yang YQ, et al. Prognostic impact of Epstein-Barr virus (EBV)-DNA copy number at diagnosis in chronic lymphocytic leukemia. Oncotarget. 2016; 7:2135–42.

7. Visco C, Falisi E, Young KH, Pascarella M, Perbellini O, Carli G, et al. Epstein-Barr virus DNA load in chronic lymphocytic leukemia is an independent predictor of clinical course and survival. Oncotarget. 2015; 6:18653–63.

8. Baliakas P, Hadzidimitriou A, Sutton LA, Rossi D, Minga E, Villamor N, et al. Recurrent mutations refine prognosis in chronic lymphocytic leukemia. Leukemia. 2015; 29:329–36.

9. Roberts AW, Davids MS, Pagel JM, Kahl BS, Puvvada SD, Gerecitano JF, et al. Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia. N Engl J Med. 2016; 374:311–22.

10. Burger JA, Sivina M, Jain N, Kim E, Kadia T, Estrov Z, et al. Randomized trial of ibrutinib vs ibrutinib plus rituximab in patients with chronic lymphocytic leukemia. Blood. 2019; 133:1011–9.

11. Li Z, Chen X, Li L, Liu S, Yang L, Ma X, et al. EBV encoded miR-BHRF1-1 potentiates viral lytic replication by downregulating host p53 in nasopharyngeal carcinoma. Int J Biochem Cell Biol. 2012; 44:275–9.

12. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008; 111:5446–56.

13. International CLL-IPI Working Group. An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data. Lancet Oncol. 2016; 17:779–90.

14. Tarrand JJ, Keating MJ, Tsimberidou AM, O'Brien S, LaSala RP, Han XY, et al. Epstein-Barr virus latent membrane protein 1 mRNA is expressed in a significant proportion of patients with chronic lymphocytic leukemia. Cancer. 2010; 116:880–7.

15. Renouf B, Hollville E, Pujals A, Tetaud C, Garibal J, Wiels J. Activation of p53 by MDM2 antagonists has differential apoptotic effects on Epstein-Barr virus (EBV)-positive and EBVnegative Burkitt's lymphoma cells. Leukemia. 2009; 23:1557–63.

16. AlQarni S, Al-Sheikh Y, Campbell D, Drotar M, Hannigan A, Boyle S, et al. Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2. Oncogene. 2018; 37:3998–4012.