Abstract

Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by specific hybridization to complementary sequences in the 3′ untranslated region of corresponding mRNAs. Concomitant recruitment of specific multi-protein complexes results either in inhibition of mRNA translation or mRNA degradation. miRNAs are processed in a regulated multi-step process from primary transcripts into mature miRNAs by cellular components which are also at least partially involved in the process of RNA interference (RNAi). Aberrant expression of specific miRNAs has recently been described in human lymphoma and leukemia. In particular, BCR-ABL and c-MYC dependent over-expression of the polycistronic and oncogenic miR-17-92 cluster (encoding miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92) has been described in chronic myeloid leukemia (CML) cell lines, primary CD34+ cells from CML patients (Venturini et al. 2007), and in lung cancer. In BCR-ABL positive K562 cells, miR-17-92 encoded miRNAs repress luciferase activity in miRNA-specific reporter assays. In addition, lentivirus-mediated over-expression of miR-17-92 increases both cell proliferation and sensitivity to imatinib induced cell death. To analyse the function of individual miRNAs of the miR-17-92 polycistron, we generated lentivirus-based strategies to induce stable miRNA-specific loss- and gain-of function phenotypes for miR-18a, miR-19b, and miR-20a, respectively. Over-expression of miRNAs embedded within miR-30-derived sequences from an internal SFFV-LTR promoter allows isolation of K562 cells with increased miRNA expression. In contrast, expression of complementary oligonucleotides (antagomirs) from a H1 promoter located in the lentiviral 3′LTR can induce stable hypomorphic miRNA-phenotypes. In lentivirally transduced K562 cells, individual silencing of miR-18a, miR-19b, and miR-20a by the corresponding antagomirs (ant-miR-18a, ant-miR-19b, ant-miR-20a) specifically relieves miRNA-mediated reporter gene repression. Correspondingly, inhibition of miRNA-function correlates to reduced ‘miRNA’-amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals specific induction of cell proliferation by miR-18a but strong inhibition by miR-20a in K562 cells, respectively. In contrast, anti-miR-18a, but not anti-miR-19b, anti-miR-20a, or control antagomirs inhibits proliferation of K562 cells. These data demonstrate individual and complementary functions of miR-17-92 encoded miRNAs in CML and identify potential targets for specific therapeutic intervention on the miRNA level.

Author notes

Disclosure: No relevant conflicts of interest to declare.