Post-transcriptional Modifications Contribute to the Upregulation of Cyclin D2 in Multiple Myeloma

• Published in: Clinical cancer research 2016-01, Vol.22 (1), p.207-217
• Main Authors: Misiewicz-Krzeminska, Irena, Sarasquete, María E, Vicente-Dueñas, Carolina, Krzeminski, Patryk, Wiktorska, Katarzyna, Corchete, Luis Antonio, Quwaider, Dalia, Rojas, Elizabeta A, Corral, Rocío, Martín, Ana A, Escalante, Fernando, Bárez, Abelardo, García, Juan Luis, Sánchez-García, Isidro, García-Sanz, Ramón, San Miguel, Jesús F, Gutiérrez, Norma C
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; Alternative Splicing; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1 - genetics; Cyclin D1 - metabolism; Cyclin D2 - genetics; Cyclin D2 - metabolism; Cyclin D3 - genetics; Cyclin D3 - metabolism; DNA Methylation; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs - genetics; Multiple Myeloma - genetics; Multiple Myeloma - metabolism; Promoter Regions, Genetic; RNA Processing, Post-Transcriptional; RNA, Messenger - genetics; RNA, Messenger - metabolism; Translocation, Genetic; Up-Regulation
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1078-0432.ccr-14-2796

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. 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. 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. Overall, these results highlight the impact of CCND2 3'UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma.