microRNA profiling in Epstein-Barr virus-associated B-cell lymphoma

Abstract

The Epstein-Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19-26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes.

Figure 1.

Overview of small RNA and miRNA gene expression in primary B-cell lymphoma tissues obtained by deep sequencing of miRNA cDNA libraries. (A) Proportions of various classes of small RNAs detected by sequencing of EBV-negative DLBCL (total reads: 97 182) and EBV-positive DLBCL (total reads: 85 221) libraries as indicated. ScaRNA, small cajal body RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; rRNA, ribosomal RNA; tRNA, transfer RNA; RNP-associated, ribonucleo-protein complex derived RNAs; unknown, derived from unannotated/intergenic regions or sequencing artefacts. (B) Distribution of miRNA genes expressed according to their sequence counts in EBV-positive and -negative DLBCL. Shown are the numbers of unique miRNA genes plotted as a function of their expression levels as defined by a given range of sequence counts in the respective libraries of small RNAs.

Figure 2.

miRNA expression in primary DLBCL tissue detected by 454 deep sequencing. (A) Distribution of EBV-encoded miRNAs in EBV-positive DLBCL. The total count of EBV-derived miRNAs was set to 100%. No reads were obtained for ebv-miR-BHRF1-1, -2, -3 and ebv-miR-BART-15 and -20. (B) Human cellular miRNAs miR-223, miR-199a-3p, miR-424, miR-27b, miR-378 and miR-26b are upregulated in EBV-positive versus -negative tissues. (C) Human cellular miRNAs miR-20b, miR-155, miR-221, miR-222, miR-151-3p, miR-29b/c and miR-106b are downregulated in EBV positive tissues. The criteria for miRNA selection were a 2-fold change and ≥50 reads (∼0.05%) abundance in one of the analyzed libraries. MiRNA expression levels are shown as percentages of the total miRNA reads in the libraries.

Figure 3.

miR-155 and -424 expression validation in primary DLBCL and DLBCL cell lines. (A) MiR-155 and miR-424 expression validation by quantitative RT–PCR. Total RNA was extracted from three tonsil tissues, and each 10 EBV positive and eight negative DLBCL (including tissues previously used for small RNA-library generation), were used for qRT-PCR analysis. Bars indicate relative expression levels compared to normal control. *P-vlaue < 0.05, student’s t-test, paired. (B) miR-155 and -424 expression validation by northern blot in DLBCL cell lines. Total RNA of U2932 cell line and EBV-positive clones (A, B, 1, 2 and 3) was detected using miRNA-specific probes and quantified by densitometric analysis. Given numbers display relative expression levels compared to loading control U6 snRNA.

Figure 4.

Target predictions for selected mRNA targets. Schematic overview of the luciferase constructs and putative bindings sites of miR-155 and -424 to their predicted mRNA targets c-MYB, SIAH1 and LATS2. The locations of the potential binding sites are indicated by shaded boxes and by the sequence alignments depicted on the right side.

Figure 5.

Luciferase reporter assays. Firefly luciferase reporters fused to the c-MYB, SIAH1, c-SKI and LATS2-3′UTRs were cotransfected with miR-155 and -424 into HEK 293-T cells in the indicated combinations. (A) MiR-155 targets c-MYB. The proximal binding site is denoted Mut1, the distal binding site is denoted Mut2, and the mutation of both binding sites is denoted Mut1+2. (B) MiR-424 targets c-MYB. The distal binding site for is denoted Mut3, the proximal site is denoted Mut4, and the mutation of both sites is called Mut 3+4. (C) miR-424 targets SIAH1 but not LATS2. The single miR-424-binding site in the SIAH1 3′UTR was mutated and denoted SIAH1 Mut. The predicted binding site for miR-424 in the LATS2-3′-UTR was not mutated as the wt-3′UTR was not responsive to miR-424. (D) miR-155 targets c-SKI. The single binding site for miR-155 was mutated and denoted SKI Mut. The values represent at least three experiments carried out in triplicate. ns: not significant.

Figure 6.

(A) Induction of c-MYB and SIAH1 protein levels by antisense inhibition of miR-155 and -424. Lysates of the EBV-positive U2932 cell clones transfected with indicated antisense inhibitors to miR-155, -424 or a scrambled oligonucleotide as a negative control were analyzed by western blotting using anti-c-MYB or anti-SIAH1 antibody. The protein input was determined by staining for β-actin. (B) Immunhistochemical staining for SIAH1: strong membranous positivity in a high percentage of tumor cells in an EBV- (A, left) DLBCL versus low expression in an EBV+ DLBCL (B, right). (C) Overexpression of miR-424 results in increased levels of β-catenin. The EBV-positive subclones clA, clB and cl2 of the DLBCL line U2932 or were treated with control oligonucleotide or with pre-miR-424 for the expression of miR-424. The levels of β-catenin and β-actin (as input control) were determined by western-blot analysis.