Regulation of proto-oncogenic Pim-1 kinase and miR-17-92 microRNAcluster in the human leukemia cell line K562
In this thesis the regulation of the oncogenic kinase Pim-1 and the microRNA cluster miR-17-92 has been investigated using the leukemia cell line K562 as a model system. Pim-1 and miR-17-92 have been reported to be highly expressed in the context of leukemia cells as well as in other cancer cell lin...
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|Summary:||In this thesis the regulation of the oncogenic kinase Pim-1 and the microRNA cluster miR-17-92 has been investigated using the leukemia cell line K562 as a model system. Pim-1 and miR-17-92 have been reported to be highly expressed in the context of leukemia cells as well as in other cancer cell lines, albeit little is known about mechanisms leading to their overexpression. The aim of the work presented here was to evaluate the functions of Pim-1 in the promotion of tumorigenesis and to clarify its regulation, with the goal of exploring its potential for therapeutic interference. Several targets of the miR-17-92 encoded miRs have been identified in different cellular contexts, and probably more will be found in the future. Nevertheless, many open questions concerning the regulation of the cluster still remain unanswered. For those reasons, we focussed our efforts on transcriptional regulation of the cluster in K562 cells, as well as on the clusters impact on one of its targets, the cell cycle regulator p21.
High expression levels of the Pim-1 kinase are characteristic for K562 cells. At the protein level, degradation of Pim-1 is promoted by the phosphatase PP2A. Inhibition of PP2A thus increases Pim-1 levels. Within the context of this work, it was shown by our group that inhibition of PP2A by ocadaic acid (OA) leads to a temporary increase of Pim-1 followed by a complete downregulation. This was accompanied by an upregulation of p21 which under normal growth conditions is not found at the protein level in this cell line. The cellular phenotype during OA-treatment changed from proliferation to apoptosis and siRNA-targeting of p21 delayed the onset of this apoptotic response, indicating that downregulation of p21 in K562 cells contributes to their anti-apoptotic and proliferative phenotype.
Interestingly, p21 protein is not found at normal growth in K562 cells, although substantial amounts of its mRNA can be detected. This implicates a posttranscriptional silencing mechanism. Here we found that p21 is a target of miR-17-5p and miR-20a, both miRs being encoded in the miR-17-92 cluster. We could prove this in two ways. First, we cloned the p21 3-UTR into a reporter vector. Mutation of the two predicted miR-binding sites in the respective constructs revealed regulation by those miRs as inferred from an increase of reporter activity. Second, we interfered with microRNA target-binding by application of antisense molecules directed against mature miR-17-5p and miR-20a. For this, we used locked nucleic acids (LNAs), as those modified oligonucleotides bind with largely increased affinity to their complementary strands. Current AntimiR approaches mostly rely on the use of 2´-O-Methyl-RNA molecules for targeting of mature miRs, as this modification stabilizes the molecules against nucleases compared to unmodified RNA. AntimiRs are designed to be complementary over the whole length of the targeted miR (22-24 nt). Here it was established in a proof of principle experiment that LNAs of only 14 nucleotides could efficiently abolish miR-function in vivo. For this, luciferase reporter constructs containing a let-7a binding site were generated. In K562 cells let-7a is abundantly expressed, so constructs were silenced when transfected, but not so in co-transfection experiments with respective LNAs targeting let-7a. LNA-AntimiRs, 8, 10, 12 or 14 nucleotides in length, were tested, and even the 8-mer showed some let-7a-specific derepression effect. With this tool at hand, we targeted miR-17-5p and miR-20a to evaluate the effects on cellular p21 protein levels. In Western blot analysis we found an induction of p21 protein upon transfection with LNA 14-mers against miR-17-5p and miR-20a. By this experimental approach, we were able to demonstrate the importance of translational downregulation of the p21 protein in K562 cells that in combination with Pim-1 overexpression is supposed to contribute to the observed proliferative phenotype...|