Leukemia (2008) 22, 330–338 & 2008 Nature Publishing Group All rights reserved 0887-6924/08 $30.00 www.nature.com/leu ORIGINAL ARTICLE Small RNAs analysis in CLL reveals a deregulation of miRNA expression and novel miRNA candidates of putative relevance in CLL pathogenesis S Marton1, MR Garcia1, C Robello2, H Persson3, F Trajtenberg4, O Pritsch5, C Rovira3, H Naya6, G Dighiero1 and A Cayota1 1Molecular Oncology Unit, Institut Pasteur, Montevideo, Uruguay; 2Molecular Biology Unit, Institut Pasteur, Montevideo, Uruguay; 3Department of Oncology, Lund University, Lund, Sweden; 4Structural Biology Unit, Institut Pasteur, Montevideo, Uruguay; 5Protein Biophysics Unit, Institut Pasteur, Montevideo, Uruguay and 6Bioinformatics Unit, Institut Pasteur, Montevideo, Uruguay MicroRNAs (miRNAs) are a novel class of small noncoding RNA useful to segregate patients into two main groups according to molecules that regulate gene expression by inducing degrada- the mutational status of the immunoglobulin heavy-chain tion or translational inhibition of target mRNAs. There are more variable-region gene (IgV ), the expression levels of the ZAP- than 500 miRNA genes reported in the human genome, H þ 8–11 constituting one of the largest classes of regulatory genes. 70 tyrosine kinase and CD38 expression, as well as the Increasing experimental evidence supports the idea of aberrant presence of cytogenetic abnormalities, such as 11q or 17p 12 miRNA expression in cancer pathogenesis. We analyzed the deletions. Patients with clones having unmutated (NM) IgVH pattern of miRNA expression in chronic lymphocytic leukemia genes or with high expression of either CD38 or ZAP-70 and/or (CLL) cells and our results showed a global reduction in miRNA 11q or 17p deletions have frequently a rapidly fatal course expression levels in CLL cells associated to a consistent despite aggressive therapy, whereas patients with mutated (M) underexpression of miR-181a, let-7a and miR-30d. We observed overexpression of miR-155 and a set of five miRNAs that are clones or low CD38 or ZAP-70 expression and no deleterious differentially expressed between patients with different clinical cytogenetic abnormalities have an indolent course and fre- outcomes. Five novel miRNA candidates cloned from leukemic quently die by unrelated diseases. Despite recent advances in cells are reported. Surprisingly, predicted mRNA targets for CLL biology the molecular mechanism involved in its initiation these novel miRNA revealed a high proportion of targets and progression remains elusive. Recently, a novel class of small located in a small region of chromosome 1, which is frequently noncoding RNAs dubbed microRNAs (miRNAs) have been altered in human cancer. Additionally, several targets were 13,14 shared by at least two of miRNA candidates. Predicted targets identified in plants and animals. Mature miRNAs of included several genes recently described as tumor suppres- 19–24 nt in length regulate target gene expression post- sors. These data could afford new avenues for exploring transcriptionally through base pairing within 30-UTR regions of innovative pathways in CLL biology and therapy. the target messenger RNAs inducing the degradation and/or Leukemia (2008) 22, 330–338; doi:10.1038/sj.leu.2405022; translational inhibition.13,15 Although the biological functions of published online 8 November 2007 miRNAs are not yet fully understood, it has been demonstrated Keywords: CLL; microRNA; gene expression that they participate in the regulation of developmental timing, cell death, cell proliferation, fat metabolism, hematopoiesis and patterning of the nervous system.16 The finding that more than Introduction half of the known human miRNAs are located at cancer- associated regions of the genome suggested that miRNAs might play a broad role in cancer pathogenesis.17 Consistent with this B-chronic lymphocytic leukemia (CLL), the commonest leuke- 1 notion is the observed aberrant expression of miRNAs in diverse mia of Western countries is characterized by elevated numbers cancer subtypes, including Burkitt’s lymphoma,18 colorectal of circulating clonal leukemic B cells. It was classically cancer,19 lung cancer,20 breast cancer21 and glioblastoma.22 considered as resulting from accumulation of minimally self- 2,3 Increasing experimental evidence implicates miRNAs either as renewing B cells as a consequence of defective apoptosis. oncogenes or tumor suppressors.23–26 Recently, two different Most peripheral CLL cells are typically in G /G tending to be 0 1 strategies were used to profile miRNA expression in CLL.27,28 By kinetically resting, phenotypically activated and functionally a microarray approach a specific expression signature consisting anergic with reduced levels of the B-cell antigen receptor of 13 miRNAs, including miR-15a and miR-16, was associated components IgM, IgD and CD79b with most malignant B cells with relevant prognostic factors.29 Following the report of bcl-2 over expressing the antiapoptotic B-cell lymphoma 2 (Bcl-2) 30 4–6 as a physiological target of these two miRNAs, a role of miR- protein. However, recent evidence indicates that CLL is not a 15a and miR-16 in CLL pathogenesis was also proposed through static disease that results simply from accumulation of long-lived the upregulation of bcl-2. However, recent reports revealed that lymphocytes, but as a dynamic process in which cells proliferate 7 significant reduction in miR-15 and/or miR-16 expression levels and die, often at appreciable levels. CLL shows a highly in CLL was not paralleled by any significant change in bcl-2.28 variable clinical course spanning from rapidly aggressive to 5 In another recent study using a cloning-based strategy, the completely indolent behaviors. Several biological markers have aberrant expression profile of miRNAs in CLL showed that miR- been described that can predict the clinical course and are 21, miR-150 and miR-155 were significantly overexpressed.28 Microarray methods are powerful for global analysis but they Correspondence: Dr A Cayota, Molecular Oncology Unit, Institut have technical limitations when used with miRNAs due to their Pasteur, Mataojo 2020, Montevideo CP 11400, Uruguay. E-mail: [email protected] short size and the sequence similarity between family members. Received 29 August 2007; accepted 8 October 2007; published Additionally and most importantly, microarray studies are online 8 November 2007 directed toward reported miRNAs, excluding the analysis of MicroRNAs and CLL pathogenesis S Marton et al 331 unannotated candidates. Therefore, to gain insight into the role verify equal loading. The RNA was electrophoretically trans- of miRNAs in CLL, we used a cloning-based approach ferred to GeneScreen Plus (Schleicher and Schuell, NH, USA). combined with stringent northern blot assays to study small After transfer, membranes were cross-linked with 120 mJ of UV RNAs from CLL patients. Our results show the global fluctuation (Stratagene, GE Healthcare, Buenos Aires, Argentina) and baked of miRNA expression levels in CLL cells compared to normal B at 80 1C for 1 h. Oligonucleotide probes were generated by T4 cells and the identification of five novel miRNA candidates from polynucleotide kinase-mediated end labeling (New England CLL cells. The observed changes are associated with significant Biolabs, Beverly, MA, USA) using [32P]g-ATP (end-labeling overexpression of miR-155 in combination with a reduction of grade, 47000 Ci mmolÀ1; MP Biomedicals, Illkirch, Strasbourg, miR-181a, let-7a and miR-30d. Notably, the differential profile France) and were purified from unincorporated label with G-25 of miRNA expression is associated with molecular prognostic microspin columns. Filters were prehybridized at 40 1Cin factors and clinical course in CLL. modified Church’s buffer (5% sodium dodecyl sulfate (SDS), 0.5% nonfat dry milk, 1 mM EDTA in sodium phosphate buffer 0.25 M, pH 7.2). After prehybridization for 2 h, radiolabeled- Materials and methods specific probes were added and further incubated for 1 h. Membranes were washed twice at 42 1C with 2 Â sodium B-lymphocyte purification from CLL patients and chloride/sodium citrate buffer (SSC) 0.1% SDS for 20 min, 1 Â healthy donors SSC 0.1% SDS for 20 min and 0.5 Â SSC 0.1% SDS for 20 min. Samples from CLL patients and healthy donors were provided by The hybridized membranes were exposed overnight to Kodak the Hematology and Bone Marrow Transplantation Unit of the Biomax MS X-ray films. Hybridized probes were stripped from Hospital Maciel (Montevideo, Uruguay) and by the National blots by washing twice in hot 0.1% SDS in deionized water for Blood Bank, (Montevideo, Uruguay), respectively, after obtain- 15 min and reprobed up to three times. ing informed consent according to locally approved regulations. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll–Metrizoate density gradient from peripheral venous blood samples. CLL lymphocytes were further Real-time RT-PCR analysis of precursor miRNAs purified from PBMCs by negative selection using magnetic For cDNA synthesis 1 mg of total RNA was reverse transcribed beads (Dynabeads, Invitrogen, Montevideo, Uruguay) coated with Superscript II reverse transcriptase (Invitrogen) using with MoAbs according to the manufacturer’s instructions, random hexamers (Invitrogen) to prime first-strand synthesis resulting in an enriched population containing more than 95% under conditions suggested by manufacturers. Quantitative PCR CD5 þ B cells. Samples from CLL patients were segregated (qPCR) was perfomed in a LightCycler 2.0 System (Roche according to the mutational status of IgVH genes, into NM and M Applied Science, Meylan, Grenoble, France) using 1 ml of cDNA groups. in 20 ml reaction volume with 0.4 mM of each specific primer and 2 mlof10Â FastStar DNA Master SYBR Green I (Roche Molecular Biochemicals, Meylan, Grenoble, France) according Cloning of small RNAs to the manufacturer’s instructions. Primers were designed Cloning of small RNAs was performed from pooled samples according to the flanking sequences of mature miRNAs (pre- belonging to three different patients corresponding respectively miRNAs), which amplify pre-miRNAs as well as primary to the NM and the M groups as defined above.