They can be subdivided into two major groups that differ slightly with regard to seed sequences

To further investigate the relationship between miR-200c, Noxa and bortezomib-induced cell death, we went on to ectopically express a Noxa construct lacking the miR-200c target site. When Noxa was overexpressed in cells left untreated with bortezomib, only a minor effect on apoptosis could be observed. However, overexpression of Noxa potentiated the positive effect of miR-200c on bortezomib-induced apoptosis, showing that artificially maintaining high Noxa levels in cells increases the pro-apoptotic effects of miR-200c even further. In summary, these data show that miR-200c sensitizes cells to bortezomib treatment. However, at the same time it represses Noxa, which leads to an attenuated bortezomib response. In this study we identify and validate miR-200c as a regulator of the proapoptotic BH3-only member Noxa. Much is known regarding the transcriptional regulation of Noxa. Several types of cellular stress, such as DNA damage and hypoxia, lead to Noxa induction in both a p53-dependent and independent fashion. However, nothing has so far been reported concerning possible microRNA regulation of Noxa. The identification of miR-200c as a Noxa regulator was facilitated by a methodology that combines a luciferase-based screening with mining of microRNA expression data. This method is broadly applicable to the identification of other microRNA:target interactions. Obviously, other mechanisms than microRNAs exist that regulate gene expression through the 39UTR.Several recent studies have demonstrated the importance of for example RNA-binding proteins in posttranscriptional gene regulation. However, it has also been shown that in many cases there is extensive interplay between microRNAs and RNA-bindingproteins. For example, miR-16 is necessary for the regulated turnover of AU-rich element containing mRNAs by the ARE-binding protein tristetraprolin. The fact that microRNA-mediated gene repression makes up a substantial part of 39UTR-mediated regulation was substantiated in a recent report investigating the impact or shortened 39UTRs on oncogenic transformation. When isoformsofvarying39UTRlengthoftheIMP-1oncogenewereused in soft-agar colony formation assays, it was demonstrated that the shorter isoforms were more oncogenic than the longer ones. Importantly, this difference in transformation ability was mostly attributed to loss of miRNA targeting, since microRNA target site mutants yielded significantly enhanced transformation from the longer isoforms. One advantage with our method is that one is not restricted to the cell lines used in the current study and it is of course Torin 1 inhibitor straightforward to change and expand the selection of cell lines to a set that is optimal for a given target gene. Furthermore, as more expression data is emerging, especially given the amounts of information originating from the recently developed mass BAY 73-4506 755037-03-7 sequencing technologies, more and more tissues will be available for consideration. Using a set of broadly used cancer cell lines, the method allowed us to relatively quickly limit the number of possible candidates and eventually end up on a true microRNA:target interaction. The interaction was validated using a series of experiments. First, overexpression of miR-200c led to dramatically reduced Noxa levels in several cancer cell lines. Importantly, this regulation occurred both in unstressed cells and cells exposed to proteasomal inhibitors. That miR-200c directly targets the 39UTR of Noxa at a defined evolutionarily conserved site was established using luciferase assays. Finally, with the help of specific miR-200c inhibitors we could show that Noxa is normally under repression from endogenous miR-200c. The miR-200 family of microRNAs consists of 5 members expressed from two genomic locations.

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