Post-Transcriptional Modifications Explain the Overexpression of CCND2 in Multiple Myeloma
Authors: Misiewicz-Krzeminska Irena , Sarasquete Maria Eugenia , Vicente-Dueñas Carolina , Krzeminski Patryk , Wiktorska Katarzyna , Quwaider Dalia , Rojas Elizabeta A. , Corral Rocío , Corchete Luis Antonio , Martin Ana A. , Escalante Fernando , Bárez Abelardo , Garcia Juan Luis , Sanchez-Garcia Isidro , García-Sanz Ramón , San Miguel Jesus F. , Gutierrez Norma Carmen 
Published: October 2019

Abstract
CCND2 is highly expressed in most of multiple myeloma (MM) samples without CCND1 or CCND3 overexpression. D-type cyclins are highly homologous proteins and there is a growing body of evidence that the functions of the D cyclins are mostly exchangeable. During mouse development, the three D-type cyclins are expressed following an often mutually exclusive pattern and their function may be tissue-specific. Likewise in MM CCND1 or CCND2 expression is commonly mutually exclusive. The mechanisms by which CCND2 is upregulated in a set of MMs are not completely deciphered. In this study, we investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at3'UTR in CCND2 overexpression in MM.
First, we observed that ectopic transfection of MM cell lines with several miRNAs, directly targeting CCND2 3'UTR according to luciferase reporter assay, decreased the level of cyclin D2, although not in all the cell lines. This fact suggested the possible disruption of miRNA target sites. Indeed, we detected the presence of short CCND2 mRNA, both in MM cell lines and primary cells, using four different methodological approaches: qRT-PCR, Northern blot, mRNA FISH and 3’RACE PCR with product sequencing. The short CCND2 isoform was observed by qRT-PCR in the majority of patients and in all MM cell lines expressing CCND2. This finding was confirmed by Northern blot results. The abundance of each CCND2 mRNA isoform was also assessed by two-color mRNA FISH designed to discriminate the two mRNA different in length. This approach also enabled us to notice that no subpopulation of cells distinguishable by the load of one isoform with respect to another was present. The results obtained by RACE experiments in MM cell lines support the idea that changes in CCND2 3’UTR length are explained by alternative polyadenylation (APA). The functional consequences of 3’UTR shortening is the mRNA stabilization due to the loss of miRNA sites and regulatory elements located in the 3’UTR. Accordingly, the luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. The short 3’UTRs lacking miRNA-binding sites have been associated with increased expression of different genes at both the mRNA and the protein level. Here, we observed significant higher level of overall CCND2 mRNA expression in those MMs with greater abundance of the shorter 3'UTR isoform. The previous intriguing observation showing that the level of cyclin D2 increased in U266 when cyclin D1 was silenced, was confirmed by our experiments. Moreover, functional analysis showed significant mRNA shortening after CCND1 silencing, suggesting that cyclin D1 could downregulate CCND2 level by modification of polyadenylation/clavage reaction. Since CCND2 expression is undetectable in myeloma cells with t(11;14), we extended our investigation to explore if DNA methylation might play a role in abolishing CCND2 expression. We observed that the CpG island more proximal to CCND2 TSS was highly methylated in MM cell lines with t(11;14).
In summary, our results reveal that CCND2 expression in MM is mainly regulated by post-transcriptional mechanisms. Downregulation of specific miRNAs directly targeting CCND2 contributes to overexpression of CCND2 in a set of MM. Moreover, the shortening of CCND2 3'UTR by alternative polyadenylation with the consequent loss of miRNA binding sites is also participating in CCND2 upregulation. In fact, this mechanism seems to play a decisive role in the regulatory network between CCND1 and CCND2 in MM.

Disclosures
No relevant conflicts of interest to declare.