Epstein-Barr Virus Infection of Cell Lines Derived from Diffuse Large B-Cell Lymphomas Alters MicroRNA Loading of the Ago2 Complex

Abstract

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoid tumor which is occasionally Epstein-Barr virus (EBV) positive and is further subtyped as activated B-cell DLBCL (ABC-DLBCL) and germinal center B-cell DLBCL (GCB-DLBCL), which has implications for prognosis and treatment. We performed Ago2 RNA immunoprecipitation followed by high-throughput RNA sequencing (Ago2-RIP-seq) to capture functionally active microRNAs (miRNAs) in EBV-negative ABC-DLBCL and GCB-DLBCL cell lines and their EBV-infected counterparts. In parallel, total miRNA profiles of these cells were determined to capture the cellular miRNA profile for comparison with the functionally active profile. Selected miRNAs with differential abundances were validated using real-time quantitative PCR (RT-qPCR) and Northern blotting. We found 6 miRNAs with differential abundances (2 upregulated and 4 downregulated miRNAs) between EBV-negative and -positive ABC-DLBCL cells and 12 miRNAs with differential abundances (3 upregulated and 9 downregulated miRNAs) between EBV-negative and -positive GCB-DLBCL cells. Eight and twelve miRNAs were confirmed using RT-qPCR in ABC-DLBCL and GCB-DLBCL cells, respectively. Selected miRNAs were analyzed in additional type I/II versus type III EBV latency DLBCL cell lines. Furthermore, upregulation of miR-221-3p and downregulation of let7c-5p in ABC-DLBCL cells and upregulation of miR-363-3p and downregulation of miR-423-5p in GCB-DLBCL cells were verified using RIP-Northern blotting. Our comprehensive sequence analysis of the DLBCL miRNA profiles identified sets of deregulated miRNAs by Ago2-RIP-seq. Our Ago2-IP-seq miRNA profile could be considered an important data set for the detection of deregulated functionally active miRNAs in DLBCLs and could possibly lead to the identification of miRNAs as biomarkers for the classification of DLBCLs or even as targets for personalized targeted treatment.IMPORTANCE Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive tumor of lymphoid origin which is occasionally Epstein-Barr virus (EBV) positive. MicroRNAs are found in most multicellular organisms and even in viruses such as EBV. They regulate the synthesis of proteins by binding to their cognate mRNA. MicroRNAs are tethered to their target mRNAs by "Argonaute" proteins. Here we compared the overall miRNA content of the Ago2 complex by differential loading to the overall content of miRNAs in two DLBCL cell lines and their EBV-converted counterparts. In all cell lines, the Ago2 load was different from the overall expression of miRNAs. In addition, the loading of the Ago2 complex was changed upon infection with EBV. This indicates that the virus not only changes the overall content of miRNAs but also influences the expression of proteins by affecting the Ago complexes.

Keywords: Ago-IP; Argonaute; DLBCL; Epstein-Barr virus; microRNA; profiling; sequencing.

FIG 1

Western blot analysis of Ago2 immunoprecipitation. Extracts of EBV-infected and noninfected U2932 cells (A) and EBV-infected and noninfected SUDHL5 cells (B) were immunoprecipitated with Ago2-specific antibodies and isotype control antibodies, as indicated. Precipitated Ago2 protein was visualized with Ago2 antibody followed by mouse anti-rat IgG coupled to horseradish peroxidase. The blot was developed using enhanced chemiluminescence (ECL). Markers on the left side of the blot indicate the positions of molecular mass marker proteins run on the same gel (prestain marker protein mix, catalog no. P77065S; New England Biolabs). The positions of the IgG heavy and light chains stained by secondary mouse anti-rat IgG coupled to horseradish peroxidase are indicated on the right side of the blot.

FIG 2

Validation of miRNA sequencing of U2932-EBV, U2932, SUDHL5-EBV, and SUDHL5 cells by RT-qPCR. Shown are comparisons of the U2932-EBV line versus U2032 (total miRNA profile) (A), U2932-EBV versus U2932 (profile of Ago2-bound miRNAs) (B), SUDHL5-EBV versus SUDHL5 (total miRNA profile) (C), and SUDHL5-EBV versus SUDHL5 (profile of Ago2-bound miRNAs) (D). The results for RNA sequencing were compared to the values obtained by RT-qPCR. NGS, next-generation sequencing.

FIG 3

Validation of total cell and Ago2-bound miRNA sequencing by Northern blotting. (A) Total RNA isolated from the U2932-EBV and U2932 cell lines (20 µg/lane) and RNA isolated from the Ago2 complexes of the same cell lines (200 ng/lane) were analyzed by Northern blotting using the probes for miR-221-3p and miR-let7c-5p, as indicated. The loading control is shown below the blots. Note that due to the small amount of RNA loaded from the Ago2-IP, no signals were obtained for the loading control. (B) Total RNA isolated from SUDHL5-EBV and SUDHL5 cells (20 µg/lane) and RNA isolated from the Ago2 complexes of the same cell lines (200 ng/lane) were analyzed by Northern blotting using the probes for miR-363-3p and miR-423-5p, as indicated. The loading control is shown below the blots. Note that due to the small amount of RNA loaded from the Ago2-IP, no signals were obtained for the loading control. EtBr, ethidium bromide.