Deregulation of Micrornas Results in Overexpression of Oncogenic Transcription Factors Involved in Pathogenesis of CLL.

MicroRNAs play a key role in the control of translation and mRNA degradation by binding to the 3′ untranslated regions of mRNAs. Immune cell development is especially dependent on miRNA regulation like miR-155 in germinal center development. Distinct miRNA-deregulation has been recently identified in CLL, however the proposed model of miR-15/16 mediated pathogenesis remains controversial. CLL cells of 50 treatment-naïve patients and peripheral B-cells of 14 healthy donors were separated by untouched depletion and processed for isolation of at least 200 ng total RNA. A new Illumina based Bead Chip was applied using 100 ng of total RNA for reliable hybridization. Target prediction of deregulated miRNAs was performed by in-silico predictions of miRNA-target gene interactions by TargetScan and PicTar. MiRNA target candidates were analyzed for differential protein expression in healthy donor B cells versus CLL cells. The applied technology was repetitively controlled and demonstrated to reveal reliable quantitative results. Comparing CLL samples with healthy donor B cells an overall decrease of miRNAs was observed in CLL samples. In total 19 miRNA were identified to be significantly lower expressed in CLL. 7 miRNAs were overexpressed in CLL. Comparing previously published data we could reliably identify upregulation of miR- 155 and downreguation of miR-181. However, the intensively discussed deregulation of miR-15 and miR-16 could not be verified. Furthermore we identified a so far not described CLL-specific miRNA-fingerprint of 26 miRNAs. Based on this fingerprint we analyzed for miRNA targets based on target prediction. A significantly focused number of transcription factors were identified by this primary screen. We could confirm predicted over-expression of oncogenic transcription factors by immunoblotting analysis. Targeting of deregulated miRNAs to 3′UTR of target genes was assessed by luciferase reporter assays. Decreased activity of 3’UTR-reporter construct was achieved by cotransfection with synthetic miRNAs together with target 3′UTRs. Targeted mutation of putative binding sites revealed an abrogation of miRNA-mediated suppression of luciferase activity confirming specificity of miRNA-3′UTR interaction of target genes. Here we could identify significant down-regulation of miRNA in CLL, a so far not identified cluster of deregulated miRNAs led to the identification of oncogenic transcription factors as novel target genes and pathogenic pathways in CLL. A key question remains regarding the cause of down-regulation of miRNAs mainly observed in CLL. Since no genomic hotspot is apparent, an impaired processing of pre-/pri-miRNAs or suppressive transcription factor loops have to be hypothesized and investigated to reveal the regulatory role of miRNA in malignant transformation of CLL.