The Synergistic Action of the microRNA-17 Cluster with c-MYC in Aggressive Cancer Development.

Malignant lymphoma frequently shows genomic amplification of 13q31-32, which harbor c13orf25, a host of mature microRNA-17, 18, 19a, 19b, 20a, and 92–1 (Ota et al., Cancer Res 2004). It has been reported that Eμ-myc mice enforced by over-expression of the miR-17-19b, could develop tumor significantly earlier than those without the expression (He et al., Nature 2005), and that overexpression of a proto-oncogene, c-MYC can up-regulate miR-17 cluster via binding directory upstream of miR-17–92 locus (O’ Donnell et al., Nature 2005). These findings suggest that the miR-17 cluster and c-MYC synergistically contribute to cancer development. However, it is still unclear how c-myc acts synergistically in tumorigenesis with the miR-17 cluster, and which genes are essentially regulated by the miR-17 cluster. In this study, we inserted 674 or 755 bp fragments spanning a given miRNA-genomic region in a modified PMXs vector such that they are placed under the control of a LTR promoter, and thus established Rat-1 and NIH3T3 cells which stably expressed miR-17–18-19a–20a-19b-92 or miR-17–18-19a-20-19b (miR-17–92/miR-17-19b). We demonstrated that the miR-17 cluster is highly upregulated under the constitutive expression of c-myc, and that a rat fibroblast (Rat-1) stably expressing both the miR-17 cluster and c-myc (miRs+myc) shows a full-blown neoplastic transformation phenotype. Because the transformation of Rat-1 cells could be due to the suppression of tumor suppressor gene(s), we next conducted screening for predicted tumor related gene(s) by a Western blot analysis with lysate from Rat-1 transfectants. Our examined predicted tumor suppressor genes of miR-17 cluster were p130 (RBR2), PTEN, SOSC-3, Smad2, MeCp2, and TGF-β type II receptor (TβRII). Among these, we could detect significant reduced expression of TβRII protein in the miRs+myc transfectant, although no other predicted targets showed such a significant difference. The expression levels of TβRII gene in Rat-1 and NIH3T3 transfected with miRs+myc were also repressed in comparison to those of the other transfectants. These results demonstrate that a combined effect of RNA destruction and translation inhibition is used by the miR-17 cluster to silence TβRII. To further substantiate TβRII as a direct target of miR-17 cluster, we cloned its 3′UTR sequence downstream of the firefly luciferase gene (pGL3-TβRII). As a control, a construct with the mutated target sequence was also made. Transfection experiments showed a significant repression of the luciferase activity in the wild type construct in comparison to mutated TβRII, thus suggesting that TβRII is direct downstream target of the miR-17 cluster. Up-regulation of miR-17 cluster and c-myc might contribute to tumorigenesis via the down regulation of both the TβRII-Smads and the c-myc-Miz-1-p15^INK4b pathways, respectively. Furthermore, we demonstrated that aggressive B-cell lymphoma frequently represents both c-MYC rearrangement and the upregulation of the miR-17 cluster via 13q31 amplification. These results suggest that the deregulation of the miR-17 cluster and c-MYC synergistically contribute to the aggressive clinicopathological features of cancers.