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


METHODS
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.
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. National Medicines Institute, Warsaw, Poland.
Irena
I
Misiewicz-Krzeminska
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
María E
ME
Sarasquete
Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. Experimental Therapeutics and Translational Oncology Program, Instituto de Biologia Molecular y Celular del Cancer, CSIC/Universidad de Salamanca, Salamanca, Spain.
Carolina
C
Vicente-Dueñas
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Patryk
P
Krzeminski
National Medicines Institute, Warsaw, Poland.
Katarzyna
K
Wiktorska
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Luis Antonio
LA
Corchete
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Dalia
D
Quwaider
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Elizabeta A
EA
Rojas
Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. Servicio de Hematología, Hospital Universitario, Salamanca, Spain.
Rocío
R
Corral
Servicio de Hematología, Hospital Universitario, Salamanca, Spain.
Ana A
AA
Martín
Complejo Hospitalario de León, León, Spain.
Fernando
F
Escalante
Hospital Nuestra Señora de Sonsoles, Ávila, Spain.
Abelardo
A
Bárez
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Juan Luis
JL
García
Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. Experimental Therapeutics and Translational Oncology Program, Instituto de Biologia Molecular y Celular del Cancer, CSIC/Universidad de Salamanca, Salamanca, Spain.
Isidro
I
Sánchez-García
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. Servicio de Hematología, Hospital Universitario, Salamanca, Spain.
Ramón
R
García-Sanz
Clinica Universidad de Navarra, Centro de Investigaciones Medicas Aplicadas (CIMA), Pamplona, Spain.
Jesús F
JF
San Miguel
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. Servicio de Hematología, Hospital Universitario, Salamanca, Spain. normagu@usal.es.
Norma C
NC
Gutiérrez
D020413:3' Untranslated Regions; D017398:Alternative Splicing; D045744:Cell Line, Tumor; D002880:Chromosomes, Human, Pair 11; D002883:Chromosomes, Human, Pair 14; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D056742:Cyclin D2 / Q000235:genetics* / Q000378:metabolism; D056743:Cyclin D3 / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D009101:Multiple Myeloma / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014178:Translocation, Genetic; D015854:Up-Regulation
D020413:3' Untranslated Regions; D056742:Cyclin D2; D056743:Cyclin D3; D035683:MicroRNAs; D012333:RNA, Messenger; D019938:Cyclin D1