Post-transcriptional modifications contribute to the upregulation of cyclin D2 in multiple myeloma
Misiewicz-Krzeminska I (1), Sarasquete ME (2), Vicente Duenas C (3), Krzeminski P (4), Wiktorska K (5), Corchete L (6), Quwaider D (7), Rojas E (1), Corral R (8), Martin AA (9), Escalante F (10), Barez A (11), Garcia JL (12), Sanchez-Garcia I (13), Garcia-Sanz R (2), San-Miguel JF (14), Gutierrez NC (15).
(1) Hematology, Centro de Investigacion del Cancer-IBMCC (USAL-CSIC).
(2) Hematology, University Hospital of Salamanca.
(3) Experimental Therapeutics and Translational Oncology Program, Centro de Investigacion del Cancer-IBMCC (USAL-CSIC).
(4) Department of Hematology, Centro de Investigacion del Cancer-IBMCC (USAL-CSIC).
(5) Confocal Microscopy Laboratory, National Medicines Institute.
(6) Servicio de Hematología, Hospital Universitario de Salamanca.
(7) Haematology, IBSAL, IBMCC, Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC.
(8) HEMATOLOGY, HOSPITAL UNIVERSITARIO DE SALAMANCA.
(9) Hematology, Hospital Universitario de Salamanca.
(10) Hematology, Complejo Hospitalario de León.
(11) Hematology, Hospital Nuestra Señora de Sonsoles.
(12) Unidad de Investigación, Instituto Estudios Ciencias de Salud de Castilla y León-Hospital Universitario de Salamanca, Centro de Investigación del Cáncer.
(13) Instituto de Biología Molecular y Celular del Cáncer, CSIC/ Universidad de Salamanca, Experimental Therapeutics and Translational Oncology Program.
(14) Clinical & Translational Medicine, Clínica Universidad de Navarra.
(15) Hematology, Institute of Biomedical Research of Salamanca (IBSAL), University Hospital of Salamanca
Dysregulation of one of the three D cyclin genes has been observed in virtually all multiple myeloma (MM) tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma (MM) 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 MM.
Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in MM 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'RACE-PCR.
We detected the presence of short CCND2 mRNA, both in MM 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 MMs 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 MM.