Ubiquitously Expressed Micro RNA miR-16 Is Not Mutated and Not Differentially Expressed in Patients with B-CLL.

MicroRNAs (miRNAs) are a recent discovered class of conserved 21–23 nucleotide short non-coding RNAs regulating protein levels by translational inhibition and mRNA destabilization and are involved in a variety of processes including cell differentiation and apoptosis. About 68% of miRNAs are tissue specifically expressed and great attention was paid to this group of miRNAs. To assess which miRNAs are ubiquitously expressed we investigated 170 human, 68 mouse and 16 rat libraries from 26 different organ systems and cell types by cloning and sequencing a total of 332,070 small RNA sequences comprising 220,147 miRNA sequences. 39 human miRNA clusters are transcribed in at least 70% of tissues and cell types. Interestingly the only miRNA cluster detected in nearly all libraries including those of embryonic origin and 8 B-CLL patient samples are the mir-16 clusters comprising mir-16-1 with mir-15a on chr.13q14 and mir-16-2 with mir-15b on chr.3q26. Earlier reports linked a lower abundance of the mature miR-16 to loss of heterozygosity (LOH) of chr.13q14 and mutations in the precursor were implicated in B-CLL in those reports. We therefore extended our investigation of B-CLL samples. We did not find any mutation in the miR-15a-miR-16-1 gene in all patients examined (64 patients). There were also no mutations in 35 healthy controls.

Furthermore 37 of 39 patients had detectable levels of miR-16 in quantitative Northern blot analysis. Only one of the two patients with undetectable miR-16 had a positive LOH status of the 13q14 locus in 90% of cells examined by FISH while the other one had no chromosomal aberrations or deletions. Additionally, the expression level of miR-16 is not correlated with the del13q14 LOH status in our study (P = 0.43 in double sided Fisher’s exact test). We also investigated the stability and processing of a reported construct bearing a C->T mutation 7 nt 3′ of the mature miRNA in 293 cell culture. This mutation has been reported in two B-CLL patients. We could not detect any difference in miR-16 expression compared to the wild type construct. Another ubiquitously expressed miRNA miR-21 is less stably expressed and is not detectable in 7 out of 39 patient samples by quantitative Northern blotting. We conclude that the absence of miR-16 in patients with B-CLL is rare and is not due to mutations in the miR-15a-miR-16-1 gene cluster in the patients examined. Furthermore miR-16 seems not to be affected by gene dosis effects like other genes involved in the LOH of 13q14 maybe because of compensatory up regulation of the other miR-16 cluster.