Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia

Abstract

Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue-specific gene regulation. MiRs are transcribed as short hairpin precursors ( approximately 70 nt) and are processed into active 21- to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via base-pairing interactions. Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL). Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority ( approximately 68%) of CLL cases.

Fig 1.

(a) Genes within the 13q14 tumor suppressor locus in CLL and localization of miR15/16 cluster. The positions of genetic markers and the positions of genes on the map are shown. (b Top) Map of the locus with previously reported 13q14 deletions marked by horizontally striped boxes. (Middle) Map of the locus between D13S1150 and D13S272 markers. The orientation of each gene is marked by an arrow under the gene's name. Colored vertical bars mark the position of corresponding exons for each gene. (Bottom) Map of the locus between Alu18 and D13S272 markers. Bars and boxes mark the position of exons for LEU2/ALT1 and LEU1 following colors in panels above. The orange arrow marks the position of miR15 and miR16 genes. Green circles mark the positions of PCR primers used to screen somatic cell hybrid clones derived from a fusion of two independent leukemia cases (CLL-A and CLL-B). Green circles mark the positions of oligonucleotides pairs used to screen hybrids by PCR. All oligonucleotides pairs shown, as well as exon-specific primers for each exon shown, and miR15/16-specific primers were used to screen the hybrids. The green arrow represents the position of the breakpoint in CLL-B carrying a t(2;13)(q32;q14) translocation. Filled boxes represent portions of chromosome 13 present in the hybrids. The 31.4-kb deletion was present in a clone derived from CLL-A, a patient with CLL carrying a t(2;13)(q12;q13) translocation, bilateral retinoblastoma, and ulcerative colitis.

Fig 2.

(a) Analysis of miR15/16 cluster expression in human tissues by Northern blotting reveals that the two genes are ubiquitously expressed, with the highest levels in normal CD5+ cells. (b) The majority of CLL samples express lower levels of miR16 and miR15 as normal CD5+ cells. The LOH status for the presented samples is shown as: +/+, heterozygosity; +/−, LOH; −/−, homozygous deletion; NI, not informative; ND, not done. As normalization controls, we used staining with ethidium bromide of Northern gels.

Fig 3.

Correlation between miR16 levels of expression (by Northern blotting) and RARS expression (by RT-PCR) in B-CLL samples. As normalization controls, we used staining with ethidium bromide (Et Br) for Northern gels and GAPDH for RT-PCRs.