Deregulation of miRNAs by Epigenetic Silencing Disrupts Suppression of the Oncogene PLAG1 in Chronic Lymphocytic Leukemia.

Abstract 3463

Poster Board III-351

MicroRNAs play a key role in cellular regulation and if deregulated in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). Both deregulations of miRNAs as well as the identification of their functional relevant targets and regulatory circuits in CLL pathogenesis are only partly understood and remain to be elucidated.

RNAs from primary cells of 50 treatment-naïve CLL patients and peripheral B-cells of 14 healthy donors were applied to miRNA-expression profiling using bead chip technology. The majority of patients presented with Binet stage A disease and showed a favorable risk profile as assessed by clinical and molecular features. Comparing the total number of miRNA being expressed a significantly lower number of miRNA was detected in CLL compared to normal B cells. The predominance of down-regulated miRNAs in CLL cells was accompanied by highly significantly lower total number of miRNAs expressed above the detection threshold in CLL patients (19.8% vs 23.5%; p<10^-6). In CLL cells a set of 7 up- and 19 down-regulated miRNAs was identified. We could not identify significant differentially expressed miRNA in cytogenetic defined subgroups, in particular we could not detect significant deregulation of miRNAs in patients harboring del13q14. Moreover, we could not identify significant down-regulation of miR-15 and miR-16 except in one patient harboring a homozygous deletion of chromosome 13q14. However, the previous up-regulation of miR-155, a key regulator of B-cell ontogenesis, appeared to be the most prominent up-regulated miRNA in our cohort. Interestingly, we identified so far unknown down-regulation of a set of miRNAs in CLL such as miR-107, -424, -125a, -126 and -326.

Among the miRNAs being downregulated in CLL cells, 6 out of 10 miRNA promoters (miR-126, miR-139, miR-181a2/b2, miR-582, miR-107, miR-449) being examined showed gain of methylation as compared to normal B cell controls. Subsequent target prediction of deregulated miRNAs revealed a highly significant binding prediction at the 3′UTR of the pleomorphic adenoma gene 1 (PLAG1) oncogene. Luciferase reporter assays including site directed mutagenesis of binding sites revealed a significant regulation of PLAG1 by miR-181a, miR-181b, miR-107 and miR-424. While expression of PLAG1 mRNA was not affected, PLAG1 protein expression was shown to be significantly elevated in CLL cells as compared to the levels in healthy donor B cells.

In conclusion we demonstrate (I) predominant down-regulation of miRNAs in CLL, (II) identified novel deregulated miRNAs in CLL, (III) unraveled underlying epigenetic changes in loci of deregulated miRNA, (IV) applied in silico target prediction of miRNA interactions for identification of novel pathogenetic factors, and (V) identified specific interaction of deregulated miRNA with PLAG1 3'UTRs resulting in over-expression of this oncogene in CLL. Therefore, PLAG1 over-expression in CLL cells represents a novel oncogenic mechanism in CLL pathogenesis on the background of deregulation in miRNA-mediated control mechanisms.