Dan-Min Xu and Yi-Lin Kong contributed equally to this work.
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.
Quantitative reverse transcription–polymerase chain reaction and western blotting were used to quantify EBV-miR-BHRF1-1 and p53 expression in cultured CLL.
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.
This study supported the role of EBV-miR-BHRF1-1 in p53 regulation
Epstein-Barr virus (EBV) was the first virus shown to encode viral microRNAs (miRNAs, miR). Five EBV-associated miRNAs were firstly identified from EBV B95-8–infected Burkitt’s lymphoma in 2004 by Pfeffer et al. [
Our previous study showed that nearly 10.0% of newlydiagnosed chronic lymphocytic leukemia (CLL) patients had a high level of EBV-DNA viral load in whole blood [
p53 gene has a vital role in the disease-progress, clone evolution and drug-resistance for CLL patients. Until now, no therapies can fully overcome the inferior prognosis caused by p53 aberration even in the era of the emerging of so many molecular targeted drugs such as BTK inhibitor and Bcl-2 inhibitors [
We collected 97 previously-untreated CLL patients from January 2004 to December 2015. The diagnosis of CLL was based on the revised National Cancer Institution criteria [
The cell lines we used for cell function experiments
Total RNA was extracted from CLL patients’ peripheral blood mononuclear cells, cultured cell lines and lentivirus transfected cell lines. RNA (1 μg) was reverse transcribed to cDNA by using specific stem-loop reverse transcription primers, forward primers, reverse primers (
Total RNA was extracted from cultured cell lines and lentivirus transfected cell lines. RNA (1 μg) was reverse transcribed using random hexamers, and amplification with fluorescent dye SYBR Green MasterMix and primers (
Renilla-luciferase assay was performed to verify whether EBV-miR-BHRF1-1 is combined with p53 gene. The pmirGLO Dual-Luciferase miRNA Target Expression Vector is designed to quantitatively evaluate miRNA activity by the insertion of miRNA target sites 3´-UTR region of the firefly luciferase gene (luc2). EBV-miR-BRHF1-1 or negative controls (NC) were transfected together with pmirGLO vector in the 293T cell line by using lipofectamine 2000 (Invitrogen, Carlsbad, CA). The ratios of Renilla versus firefly signals was used to measure the efficiency of transfection by using dualluciferase reporter gene assay kit (Promega, Madison, WI) at 24 hours and 48 hours after transfection.
Cells were harvested after stable transfection with miRNA inhibitors (including EBV-miR-BHRF1-1 and NC miRNA inhibitors) and miRNA mimics (including EBV-miR-BHRF1-1 and NC miRNA mimics). CLL patients’ peripheral blood mononuclear cells and cells of transfection were and solubilized by incubating in ice-cold RIPA lysis buffer. Then cells were fragmented fully by ultrasonic instruments. Protein concentration was determined by BCA (Real-Times, Shanghai, China). Proteins were added in sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) loading buffer (Beyotime, Shanghai, China) for dye and storage. Protein samples separated on 12% SDS-PAGE (Beyotime), transferred to polyvinylidene difluoride membranes (Immobilon-P Membrane) followed by incubating with a mouse monoclonal antibody to p53 (Cell Signaling Technology, Boston, MA) at 1:1,000 dilutions, a rabbit monoclonal antibody to p-p53 (Cell Signaling Technology) at 1:1,000 dilutions, a rabbit monoclonal antibody to PDLIM7 (Cell Signaling Technology) at 1:1,000 dilutions, and a rabbit monoclonal antibody to p21 (Cell Signaling Technology) at 1:1,000 dilutions. The secondary antibody used was rabbit anti-goat IgG (Multi Sciences) and mouse anti-goat IgG (Multi Sciences) both at 1:3,000 dilutions at room temperature. Protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology), a mouse monoclonal antibody, at 1:10,000 dilutions. Antibody complexes were detected by enhanced chemiluminescence (Merck Millipore, Darmstadt, Germany).
Cells were plated in 96-well plates at a density of 1.0×104 cells/well with 100 μL IMDM medium, and stabilized cells for 4 hours at 5% CO2, 37°C incubator (Thermo Fisher Scientific, Boston, MA). Then take out to add in 10 μL Cell Counting Kit-8 (CCK-8) at once, 24, 48, 72, and 96 hours after planked cells in triplicate, using CCK-8 kit (Dojindo, Kumamoto, Japan) and incubate cells continuously. After 2 hours, OD value was tested by using a microplate reader (Bio-Tek, Winooski, VT) and data were collected by Gene5 software (Bio-Tek).
Cells after stable transfection was performed to detect apoptosis ratio by using Annexin V propidium iodide (PI)/ 7-aminoactinomycin D (7-AAD) flow cytometric assay kit (BD Pharmingen, Franklin Lakes, NJ). We collected 1.0×106 transfected cells into a tube, and cells were washed twice with cold phosphate-buffered saline (PBS) and re-suspended in 1 mL Annexin V binding buffer. Cells were incubated in the dark for 15 minutes at room temperature after the addition of 5.0 μL of PI and 3.0 μL of 7-AAD. We added 4 mL binding Annexin V buffer additionally before test. Apoptosis of lentivirus transfected cells was quantified using a FACSCalibur flow cyto-meter (BD FACSCanto II Flow Cytometer) and FlowJo software. Phycoerythrin and PI are the same fluorescent emission channel. 7-AAD and PerCP-Cyanine5- 5 (PerCP-cy5-5) are the same fluorescent emission channel. Experiments were done in triplicate.
Cells at the concentration of 1.0×106 were incubated and saved at –20°C for 30 minutes fixed in 70% ethanol, then washed twice in PBS and incubated in 1 mL DNA staining solution and 10 μL PI for 30 minutes at 37°C using cell cycle staining kit (MultiSciences). DNA content was determined by flow cytometry using Beckman Coulter Gallios flow cytometry (Boston, MA) and analyzed by Wincycle 32 software (Beckman Coulter). Experiments were done in triplicate.
Statistical analyses were performed using SPSS software for Windows ver. 20.0 (IBM Corp., Armonk, NY). The difference of target gene mRNA expression between groups with different prognostic factors was described using the MannWhitney U test. The difference of miRNA and p53 expression and apoptosis rate between groups was described using the paired-samples T test. The receiver operating characteristic (ROC) curve was used to determine the cut-off value. OS was calculated as time from diagnosis until death or last followup. TTT was calculated as interval from diagnosis until the first CLL-specific treatment or the last follow-up. Survival and TTT were estimated by the Kaplan-Meier method and results were compared using the log-rank test. The prognostic influence of variables was tested using the Cox proportional hazards model in univariate and multivariate analysis. Protein bands of Western blot were quantified through the Image J program for Windows after normalizing the data for GAPDH. Two-sided p-values of < 0.05 were considered statistically significant.
Subject gave informed consent in accordance with requirements of the Declaration of Helsinki. The research was approved by the Institutional Review Boards of Nanjing medical University.
The baseline clinical and biological variables of the 97 newly-diagnosed CLL patients are shown in
The relative expression level of EBV-miR-BHRF1-1 in all subjects was 0.00001 to 0.05120 (median, 0.01056). EBV-miRBHRF1-1 expression of CLL subjects was analyzed for association with baseline variables (
ROC curve analysis of OS was used to determine the optimal threshold cut-off value for EBV-miR-BHRF1-1 level. Area under the curve was 0.651 (95% confidence interval [CI], 0.531 to 0.771; p < 0.001). The optimal cut-off value of EBV-miR-BHRF1-1 was 0.0012, with a 51% sensitivity and a 77% specificity. We split the cohort of CLL into low (n=49) and high (n=48) EBV-miR-BHRF1-1 expression based on a value of 0.0012. Subjects in the low EBV-miR-BHRF1-1 expression cohort had a briefer TTT (p=0.018) and worse OS (p < 0.001) compared with subjects in high EBV-miR-BHRF1-1 expression cohort (
In univariate analyses, Binet B/C stage, ALC ≥ 50×109/L, LDH > ULN, EBV-DNA > 5,000 copies/mL,
In univariate analyses, LDH > ULN,
The computational algorithms, including TargetScan and RNAhydrid, were used in combination to identify potential miRNAs that target p53. As shown in
To further investigate whether there is also a negative correlation between EBV-miR-BHRF1-1 levels and p53 expression in primary EBV-positive CLL cases, EBV-miR-BHRF1-1 and p53 expression were assessed in five CLL cases (
We then explored the biological effects of inhibiting EBV-miR-BHRF1-1 on cell lines of MEC1 and JVM3 and whether of these effects may be mediated through increase of p53 levels. We only used EBV-miR-BHRF1-1 inhibitor to decrease the EBV-miR-BHRF1-1 levels in cell lines. qRT-PCR, western blotting (WB) and cell function tests were conducted after cells were transfected stable. Significant reductions of EBV-miR-BHRF1-1 expression level were observed in both MEC1 and JVM3 (
We then explored the biological effects of overexpression of EBV-miR-BHRF1-1 on cell lines of MEC1 and JVM3 and whether these effects may be mediated through decrease of p53 levels. We only used EBV-miR-BHRF1-1 mimic to increase the EBV-miR-BHRF1-1 levels in cell lines. qRT-PCR, WB and cell function tests were conducted after stably transfected. A significant increase of EBV-miR-BHRF1-1 expression level was observed in JVM3 (
In this study, we show that high expression levels of EBV-BHRF1-1 in CLL patients were predictive of inferior TTT and OS. The predictive power of OS was retained after adjusted for all the prognostic factors in International Prognostic Index for patients with CLL [
Thought the clues of EBV in CLL patients have been reported in several studies, we still did not know the latency type of EBV in EBV-positive CLL patients. Combined with the facts that LMP1 mRNA reported by Tarrand et al. [
The study reported by Li et al. [
In conclusion, we presented evidence that p53 is a target of EBV-miR-BHRF1-1, which might be the underlying pathologic mechanisms for the inferior prognostic value for high expression levels of EBV-miR-BHRF1-1 in patients with CLL. The interaction between EBV-miR-BHRF1-1 and p53 may represent one of the many ways by which EBV-miR-BHRF1- 1 promotes EBV-positive lymphomagenesis of latency III type. Our results support the potential of EBV-miR-BHRF1- 1 as a therapeutic target for CLL patients with p53 gene aberrations.
Supplementary materials are available at Cancer Research and Treatment website (
Sequences of qRT-PCR primers of EBV-miR-BHRF1-1 and p53
Conflict of interest relevant to this article was not reported.
This study was supported by National Natural Science Foundation of China (81470328, 81600130, 81770166, 81720108002), Jiangsu Province’s Medical Elite Programme (ZDRCA2016022), Project of National Key Clinical Specialty, Jiangsu Provincial Special Program of Medical Science (BE2017751) and National Science and Technology Major Project (2018ZX09734007).
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
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).
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.
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.
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.
Clinical variables and associations between EBV-miR-BHRF1-1 and baseline variables for the 97 CLL subject
Value (%) | Median (10–5) (95% CI) | p-value | |
---|---|---|---|
≤ 65 | 64 | 129.0 (1.251485-4,867.529) | 0.501 |
> 65 | 33 | 121.0 (1.75156-1,670.395) | |
Male | 67 | 148.0 (1.251485-4,867.529) | 0.833 |
Female | 30 | 97.5 (1.75156-1,670.395) | |
A | 34 | 122.0(1.251485-727.867) | 0.506 |
B/C | 63 | 121.0 (1.554705-4,867.529) | |
< 50 | 66 | 119.5 (1.251485-4,867.529) | 0.996 |
≥ 50 | 31 | 140.0 (1.75156-4,403.11) | |
≤ ULN | 66 | 103.0 (1.251485-4,867.529) | 0.007 |
> ULN | 31 | 409.0 (1.75156-4,727.867) | |
≤ ULN | 33 | 193.2 (1.554705-4,867.529) | 0.651 |
> ULN | 64 | 117.3 (1.251485-4,727.867) | |
≤ 5,000 | 76 | 82.53 (1.251485-4,155.982) | < 0.001 |
> 5,000 | 21 | 577.4 (21.70026-4,867.529) | |
Mutation | 58 | 110.8 (1.75156-4,867.529) | 0.378 |
Unmutation | 39 | 197.2 (1.251485-4,403.11) | |
No | 74 | 82.53 (1.251485-4,867.529) | < 0.001 |
Yes | 23 | 409.2 (21.64018-4,403.11) |
EBV, Epstein-Barr virus; CLL, chronic lymphocytic leukemia; CI, confidence interval; LDH, lactate dehydrogenase; β2-MG, β2-microglobulin.
Univariate and multivariate Cox regression analysis for TTT and OS in patients with CLL
Variable | Univariate analysis to TTT |
Multivariate analysis to TTT |
Univariate analysis to OS |
Multivariate analysis to OS |
||||
---|---|---|---|---|---|---|---|---|
HR (95% CI) | p-value | HR (95% CI) | p-value | HR (95% CI) | p-value | HR (95% CI) | p-value | |
Age > 65 yr | 1.068 (0.650-1.754) | 0.795 | - | - | 0.815 (0.306-2.537) | 0.882 | - | - |
Male sex | 1.275 (0.745-2.184) | 0.376 | - | - | 0.985 (0.342-2.835) | 0.978 | - | - |
Binet B/C stage | 2.817 (1.614-4.916) | < 0.001 | 1.413 (0.835-2.390) | 0.197 | 1.619 (10.52-5.020) | 0.404 | - | - |
ALC ≥ 50×109/L | 2.323 (1.409-3.829) | 0.001 | 1.960 (1.068-3.599) | 0.030 | 1.277 (0.464-3.515) | 0.635 | - | - |
LDH > ULN | 2.138 (1.303-3.509) | 0.003 | 1.695 (1.009-2.848) | 0.046 | 2.737 (1.019-7.350) | 0.046 | 1.361 (0.493-3.762) | 0.552 |
β2-MG > ULN | 1.471 (0.879-2.461) | 0.142 | - | - | 0.859 (0.312-2.365) | 0.769 | - | - |
EBV-DNA > ULN | 2.020 (1.171-3.484) | 0.012 | 1.191 (0.632-2.243) | 0.588 | 4.653 (1.733-12.494) | 0.002 | 1.528 (0.500-4.665) | 0.457 |
IGHV unmutated | 2.556 (1.569-4.164) | < 0.001 | 1.828 (1.083-3.087) | 0.024 | 5.919 (1.687-20.770) | 0.006 | 4.433 (1.223-16.069) | 0.023 |
p53 disruption | 1.495 (1.085-2.060) | 0.014 | 1.093 (0.735-1.626) | 0.661 | 2.328 (1.343-4.035) | 0.003 | 2.316 (1.197-4.482) | 0.013 |
EBV-miR-BHRF1-1 high expression | 1.676 (1.036-2.711) | 0.035 | 1.492 (0.884-2.516) | 0.134 | 7.146 (1.624-31.441) | 0.009 | 5.335 (1.193-23.846) | 0.028 |
TTT, time-to-treatment; OS, overall survival; CLL, chronic lymphocytic leukemia; HR, hazard ratio; CI, confidence interval; ALC, absolute lymphocyte level; LDH, lactate dehydrogenase; ULN, upper limit of normal; β2-MG, β2-microglobulin; EBV, Epstein-Barr virus; miR, microRNA.