MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias

Abstract

Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673-676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253-258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia.

Fig. 1.

Two distinct miRNA signatures characterize the CLL samples. Thirty-eight CLL patients were analyzed; from three of them two different cell samples were processed and analyzed. In all instances, the different samples from the same patients clustered together. The main miRNA-associated CLL clusters are presented. The control samples are: MNC; Ly, lymph node; CD5+, selected CD5+ B lymphocytes.

Fig. 2.

Microarray data confirmation. (A) Northern blots of CLL RNAs showing concordance with the microarray data. Names of miRNA and specific oligonucleotides spotted on the array are presented. The numbers correspond to absolute expression value of each miRNA (determined by a per-chip on median normalization) and, therefore can be compared with the band intensity on Northern blots. 5S RNA stained with ethydium bromide (Et-Br) was used as loading control. The significance of the two oligos designed for some miRNAs is described in Tables 4–6. Generally, the oligoNo1 corresponds to the active part, whereas the oligoNo2 corresponds to the precursor miR molecule. PBL, peripheral blood leukocytes. (B) Real-time PCR quantification of miRNA expression in MNC and CLL samples. Expression of miR-15a (Left) and miR-30d (Right) by microarray (yellow bar) is compared with the levels detected with the two oligo specific for the active part (red bar) or precursor miRNA (blue bar). Correlations only with the expression levels of pre-miRNA (blue bar) but not active miRNA (red bar) were found. The data represent the mean of triplicate real-time PCRs from a single DNA sample. (C) Variations in the expression of Pten protein, a target of miR-19a are paralleled, in six of seven CLLs, by miR-19a microarray expression. The numbers represent MeCP2, the methyl-CpG-binding protein excluded as a putative target of miR-19a was used as the negative control. The numbers correspond to absolute expression value of miR-19a (determined by a per-chip on median normalization). Actin was used as loading control.