Regulation of the Cyclin D1 Proto-Oncogene by Cell Cycle Regulatory MicroRNAs.

Mutations, activation or overexpression of cyclin D1 are common features of several human cancers including mantle cell lymphoma (MCL) which bears the characteristic t(11;14) translocation juxtaposing the cyclin D1 gene downstream of the immunoglobulin heavy chain enhancer. The loss of the 3’UTR of this gene has been reported in a majority of MCL patients as well as in cell lines. In order to assess the impact of the 3’UTR on cyclin D1 expression levels, we used YFP tagged cyclin D1 reporter plasmids to quantify cyclin D1 expression in cell lines with different mutations of the 3’UTR. Interestingly, protein expression was significantly higher upon deletion of the cyclin D1 3’UTR compared to the full-length cyclin D1 gene as assessed by flow cytometry (2.1 fold; n=3, P < 0.05). Applying a more sensitive dual-luciferase reporter assay where a constitutively expressed luciferase gene was fused to the cyclin D1 3’UTR, the normalized firefly luciferase activity was reduced significantly to 23% as compared to luciferase only (the empty vactor). We then introduced 3’UTR mutations observed in MCL patients (insertion of adenosine between nucleotides 2308 and 2309 and a deletion of the tri-nucleotide sequence TCA from 2309–2311 of the full length cyclin D1-YFP reporter cDNA), which resulted in a significant increase of cyclin D1 expression (1.3 fold both in Ins308 and Δ309-311) compared to full length cyclin D1, (P< 0.05) showing that these mutations contribute to cyclinD1 overexpression in these patients. Subsequently, the 3’UTR was scanned for elements potentially regulating cyclin D1 expression, and putative microRNA binding sites were identified using the TargetScan and PicTar microRNA target prediction software. The most interesting candidate microRNAs include the miR-15/16 family and the miR-17–92 cluster, both of which have been shown to be involved in lymphoid malignancies and regulate cell cycle progression. In order to confirm whether the cyclin D1 3’UTR is a direct target of these microRNAs, we cloned the cyclin D1 3’UTR target region containing putative miR-15/16 or miR-17/20a binding sites and transfected these reporter constructs into HeLa cells. Upon introduction of oligonucleotide mimics of the miR15/16 microRNAs or a plasmid expressing microRNAs of the miR-17 cluster, the normalized luciferase activity of the respective luciferase reporters was reduced significantly to 41% (miR-15), 33% (miR-16) and 79% (miR-17/20a), respectively. Moreover, introduction of mutations in the seed sequences of the putative microRNA recognition sites rendered these constructs insensitive to inhibition by these microRNAs, confirming the specificity of the microRNA::target interaction. These data confirm that the binding of these microRNAs play an important role in the repression of cyclin D1 mediated by the 3’UTR and mutation or deletion result in cyclin D1 overexpression in MCL as well as other human tumors.