CircEAF2 counteracts Epstein-Barr virus-positive diffuse large B-cell lymphoma progression via miR-BART19-3p/APC/β-catenin axis

Abstract

Background: Epstein-Barr virus (EBV) represents an important pathogenic factor of lymphoma and is significantly associated with poor clinical outcome of diffuse large B-cell lymphoma (DLBCL). Circular RNAs (circRNAs) play an essential role in lymphoma progression. However, the underlying mechanism of circRNA on DLBCL progression related to EBV remains largely unknown.

Methods: CircRNA was screened by high-throughput sequencing in tumor samples of 12 patients with DLBCL according to EBV infection status. Expression of circEAF2, as well as the relationship with clinical characteristics and prognosis, were further analyzed in tumor samples of 100 DLBCL patients using quantitative real-time PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of circEAF2 both in vitro and in vivo. The underlying mechanism of circRNA on DLBCL progression were further determined by RNA sequencing, RNA pull down assay, dual-luciferase reporter assay, rescue experiments and western blotting.

Results: We identified a novel circRNA circEAF2, which was downregulated in EBV + DLBCL and negatively correlated with EBV infection and DLBCL progression. In EBV-positive B lymphoma cells, circEAF2 overexpression induced lymphoma cell apoptosis and sensitized lymphoma cells to epirubicin. As mechanism of action, circEAF2 specifically targeted EBV-encoded miR-BART19-3p, upregulated APC, and suppressed downstream β-catenin expression, resulting in inactivation of Wnt signaling pathway and inhibition of EBV + DLBCL cell proliferation. In EBV-positive B-lymphoma murine models, xenografted tumors with circEAF2 overexpression presented decreased Ki-67 positivity, increased cell apoptosis and retarded tumor growth.

Conclusions: CircEAF2 counteracted EBV + DLBCL progression via miR-BART19-3p/APC/β-catenin axis, referring circEAF2 as a potential prognostic biomarker. Therapeutic targeting EBV-encoded miRNA may be a promising strategy in treating EBV-associated lymphoid malignancies.

Keywords: Diffuse large B-cell lymphoma; Epstein-Barr virus; Wnt signaling pathway; circEAF2; miR-BART19-3p; β-catenin.

Fig. 1

Identification and characterization of circEAF2 in Epstein-Barr virus-positive diffuse large B cell lymphoma. A Volcano plot showed the differential expressed circRNAs of EBV + DLBCL (N = 8), as compared to EBV-DLBCL (N = 4), analyzed by circRNA high-throughput sequencing. B Relative expression of circEAF2 from 7 B-lymphoma cell lines and 54 DLBCL tumor samples. Results on tumor samples was further transformed by LogE. C Relative expression of circEAF2 (line graph) and EBV DNA copy number (bar graph) in SU-DHL-4 and OCI-LY-10 cells during acute EBV infection or long-term EBV infection, as compared to those without EBV infection (negative control, NC). D Schematic diagram showed the genomic locus of circEAF2 in EAF2 gene. Sequencing results showed the back-splice junction sequences of circEAF2. Arrows represented divergent primers that bind to the back-splicing region of circEAF2. E Left panel: The expression of circEAF2 and mRNA EAF2 after RNase R treatment. Right panel: The expression changes of circEAF2 and mRNA EAF2 at the corresponding time points after treatment with amphotericin D. Results are presented as the RNA expression level relative to those without RNase R or amphotericin D treatment (negative control, NC). F qRT-PCR assay with divergent or convergent primers indicated the existence of circEAF2 or mRNA EAF2, respectively, using cDNA and gDNA as templates. EBV, Epstein-Barr virus; DLBCL, diffuse large B-cell lymphoma; circRNA, Circular RNA; EBV+, EBV positivity; EBV-, EBV negativity; R, Reverse primer; F, Forward primer; gDNA, Genomic DNA; cDNA, Complement deoxyrubonucleic acid. Con, control group. Data were shown as the mean ± S.D. of three experiments

Fig. 2

CircEAF2 is associated with EBV infection and clinical outcomes in DLBCL patients (N = 100). A Relative expression of circEAF2 according to tumor EBER-ISH, tumor EBV DNA or serum EBV DNA of DLBCL patients. Results on tumor samples was further transformed by LogE. B Correlations of circEAF2 expression with EBV DNA copy number in tumor and in serum. C Progression-free survival (PFS) and overall survival (OS) of DLBCL patients according to circEAF2 expression. D Forest plot showed univariate and multivariate prognostic analysis for PFS and OS of DLBCL patients. EBER, EBV-encoded RNA; ISH, in-situ hybridization; PFS, Progression-free survival; OS, overall survival; HR, Hazard ratio; CI, Confidence interval; LDH, Lactate dehydrogenase; IPI, International prognostic index; ECOG, Eastern Cooperative Oncology Group; CR, Complete remission; GCB, Germinal center B-cell

Fig. 3

CircEAF2 counteracts the progression of EBV + DLBCL in vitro. A CircEAF2 overexpression in Farage cells and circEAF2 silencing in SU-DHL-4 cells. B CCK8 assay evaluated cell proliferation at each time point. C and D Annexin V-APC apoptosis (C) and TUNEL assay (D) detected lymphoma cell apoptosis (ratio). E Dose-response curves and the half maximal inhibitory concentration (IC50) for epirubicin on circEAF2-overexpressing cells or circEAF2-slienced cells. Scale bar represented 200 μm. Si-NC, Negative control siRNA; Si-circEAF2, circEAF2 siRNA; TUNEL, Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole; IC50, Half maximal inhibitory concentration. Data were shown as the mean ± S.D. of three experiments

Fig. 4

CircEAF2 functions as a sponge of miR-BART19-3p in EBV + DLBCL. A CircEAF2-interacting miRNAs profiles were pulled by the biotinylated-circEAF2 probe and compared with oligo probe. B The binding ability of miRNAs was further confirmed by circEAF2 luciferase reporter. The groups transfected wild-type circEAF2 luciferase reporter without miRNA mimics and scramble mimics were used as control group (Ctrl) and negative control group (NC), respectively. C Left panel: Schematic model showed the putative-binding sites for miR-BART19-3p and circEAF2. Right panel: Luciferase assay was performed in HEK293T cells transfected with the wild-type or mutant circEAF2 luciferase reporter and the indicated concentrations of miR-BART19-3p mimics. Results were normalized to a Renilla transfection control. D Enrichment of circEAF2 was pulled down by biotinylated-miR-BART19-3p. E Overexpression of miR-BART19-3p in Farage and SU-DHL-4 cells. Cell proliferation (F), apoptosis assay (G), and epirubicin sensitivity (H) were performed after co-transfection of miR-BART19-3p mimics with circEAF2 plasmid in Farage cells or circEAF2 siRNA in SU-DHL-4 cells. miR-BART19-WT, miR-BART19-3p mimics; miR-BART19-Mut, miR-BART19-3p mutants; Data were shown as the mean ± S.D. of three experiments

Fig. 5

CircEAF2 functions as a sponge of miR-BART19-3p in EBV + DLBCL. A Gene set enrichment analysis (GSEA) according to circEAF2 expression. B Correlation of miR-BART19-3p expression with circEAF2 expression, APC expression with circEAF2 expression, APC expression with miR-BART19-3p expression in DLBCL tumor samples. Results on tumor samples was further transformed by LogE. C Left panel: Schematic model showed the putative-binding sites for miR-BART19-3p and circEAF2. Middle panel: Luciferase assay was performed in HEK293T cells transfected with the wild-type or mutant APC luciferase reporter and the indicated concentrations of miR-BART19-3p mimics. Right panel: APC-3’UTR luciferase reporter assay was evaluated in HEK293T cells co-transfected with mutant circEAF2 and miR-BART19-3p mimics, or with indicated concentrations of wild-type circEAF2 and miR-BART19-3p mimics. D APC-3’UTR luciferase reporter assay was evaluated in Farage cells co-transfected with wild-type circEAF2 and with miR-BART19-3p mimics. Results were normalized to a Renilla transfection control. E Relative mRNA expression of APC in Farage cells transfected with wild-type circEAF2 and miR-BART19-3p mimics. F Immunoblot assay of APC and nuclear β-catenin proteins in Farage cells transfected with wild-type circEAF2 and miR-BART19-3p mimics. Numbers showed quantification of relative protein amount. GAPDH was used as the control of total protein. Histone3 was used as the endogenous control for nuclear protein. G Schematic model showed the regulator network of circEAF2/miR-BART19-3p/APC/β-catenin in EBV + DLBCL. Created with BioRender.com. GSEA, Gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes. Data were shown as the mean ± S.D. of three experiments

Fig. 6

CircEAF2 attenuates the progression of EBV + DLBCL in vivo. A In the untreated and epirubicin group, the growth curve of xenograft tumors transfected with circEAF2 overexpression and with vector. B Immunohistochemistry analysis of Ki-67 and TUNEL assay of cell apoptosis in xenograft tumors transfected with circEAF2 overexpression and with vector. HE, hematoxylin-eosin staining. Untreated, the untreated group; Epirubicin, the epirubicin group; Scale bar represents 200 μm. Data were shown as the mean ± S.D. of three experiments