Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

Abstract

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project "Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing", we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.

Figure 1.

The miRnome of B-cell lymphomas. (A) Clustering according to the top 25 differentially expressed miRNA inferred with edgeR between FL (light blue), DLBCL (blue), and BL (gray), in pairwise comparisons. (B) Clustering according to the top 25 differentially expressed miRNA inferred with edgeR between BL and DLBCL/FL. (C) Hsa-miR-143 expression across all patients’ samples. (D) Visualization of the genomic mutations of those miRNA, which show alterations in their mature sequences. The mature sequences of the respective miRNA are shown; the seed sequences are highlighted by black boxes. The positions of the mutations are also indicated. (E) Predicted folding of the three biochemically validated novel miRNA.

Figure 2.

Direct miRNA-mRNA regulation in B-cell lymphomas. (A) PAR-CLIP principle. Following the addition of 4-thiouridine, an immunoprecipitation with subsequent protein digestion is performed. Purified RNA fragments are reverse transcribed and cDNA libraries are sequenced on a HiSeq2500 followed by bioinformatic analysis (adapted from Hafner et al.). (B) PAR-CLIP library statistics. The left y-axis shows the number of aligned reads, the right y-axis the number of high quality PAR-CLIP clusters. The cell lines employed are indicated. (C) Flow chart of the integrative miRNA-mRNA regulation analysis (adapted from Farazi et al.). (D) List of lymphoma relevant genes for which regulation by distinct miRNA could be elucidated.