Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. National Medicines Institute, Warsaw, Poland.
Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Clin Cancer Res. 2016 Jan 1;22(1):207-17. doi: 10.1158/1078-0432.CCR-14-2796. Epub 2015 Sep 4.
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.
几乎所有多发性骨髓瘤肿瘤中都观察到三个 D-细胞周期蛋白基因之一的失调。在一组多发性骨髓瘤中，CCND2 上调的机制尚未完全阐明。我们研究了通过 miRNA 与其在 3'UTR 上的结合位点之间的相互作用进行的转录后调控在多发性骨髓瘤中 CCND2 过表达中的作用。
本研究纳入了 11 个骨髓瘤细胞系和 45 个原发性骨髓瘤样本。通过 3'UTR 荧光素酶质粒测定分析多发性骨髓瘤中失调的 miRNA 与 mRNA 靶标的相互作用。使用 qRT-PCR、Northern blot、mRNA FISH 和 3'快速扩增 cDNA 末端（RACE）-PCR 探索长度不同的 CCND2 mRNA 异构体的存在。
我们在多发性骨髓瘤细胞系和原代细胞中均检测到短的 CCND2 mRNA 的存在。3'RACE 实验结果表明，CCND2 3'UTR 长度的变化是由可变多聚腺苷酸化解释的。含有截断 CCND2 mRNA 的质粒的荧光素酶测定强烈证实了较短的 CCND2 mRNA 异构体中 miRNA 位点的缺失。那些较短 3'UTR 异构体丰度较高的多发性骨髓瘤与总 CCND2 mRNA 表达水平显著升高相关。此外，功能分析表明 CCND1 沉默后 CCND2 mRNA 明显缩短，CCND1 和 CCND3 过表达后较长异构体的相对表达增加，表明细胞周期蛋白 D1 和 D3 可通过多聚腺苷酸化切割反应的修饰调节 CCND2 水平。
总体而言，这些结果强调了 CCND2 3'UTR 缩短对多发性骨髓瘤中 miRNA 依赖性 CCND2 调节的影响。
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