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Purpose: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma.Experimental Design: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR.Results: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction.Conclusions: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma. Clin Cancer Res; 22(1); 207–17. ©2015 AACR.

Supplementary Tables Supplementary Table 1. Cytogenetic characteristics of MM samples Supplementary Table 2. Sequences of primers used in the study Supplementary Table 3. CCND2 mRNA FISH probes sequences Supplementary table 4. microRNAs predicted to target CCND1 or/and CCND2 Supplementary table 5. microRNAs expression in MM cell lines. Supplementary Figures Supplementary Figure 1. CCND2 mRNA level 24h after transfection of MM1S, RPMI and H929 cell lines with indicated miRNAs Supplementary Figure 2. Expression of miR-15a and miR-196b in H929 and MM1S cell lines after transfection Supplementary Figure 3. (A) Alignment of 3'RACE PCR product sequence from MM1S cell line with CCND2 mRNA (NM_001759.3). (B) Sequence of the 3'RACE PCR product withhighlighted APA signal. Supplementary Figure 4. CCND2 mRNA expression visualized with FISH probes Supplementary Figure 5. Luciferase activity in HEK923 cells cotransfected with various miRNAs, plasmid encoding GFP and plasmid containing luciferase and 3'UTR of CCND2 short isoform Supplementary Figure 6. Regulation of cyclin D2 level by CCND1/CCND3.