Oncogenic role of the non-canonical base-pairing between miRNAs/mRNAs in uveal melanoma.

Chromosome 3p monosomy is associated with a poor clinical outcome of patients with uveal melanoma. Since a copy of the tumor suppressor miR-16 gene is lost for these patients, we postulated that a 3p loss may reduce the miR-16 amount and activity, promoting RNA derepression and tumor burden (loss of brake effect) as observed in chronic lymphocytic leukemia. Unexpectedly, we found that miR-16 expression level is not decreased despite the 3p monosomy. In contrast, our results suggested that miR-16 activity is impaired in uveal melanoma. Here, we investigated the molecular mechanism explaining the sequestration of miR-16 by RNAs. By defining the miR-16 interactome, two genes sets have been highlighted, suggesting two divergent miR-16 functions. In addition to the canonical miR-16 targets such as CCND3 and CDC25A, we identified another set of miR-16-interacting RNAs called thereafter miR-16 sponges. miR-16 binds to these RNAs sponge without inducing their decay. Mechanistically, the miR-16/RNA non-canonical base-pairing promoted stability of mRNAs involved in cancer cell proliferation. The biological relevance has been challenged in uveal melanoma. We showed that patients with poor overall survival expressed higher levels of miR-16 sponges and canonical miR-16 targets. These results strongly suggested that miR-16 is no longer able to repress its targets and, in contrast, promotes RNA stability and protein expression of oncogenes. miR-16 activity assessment using our Sponge-signature discriminates the patient’s overall survival as efficiently as the current method based on copy number variations and driver mutations detection. To conclude, miRNA loss of function due miRNA sequestration seems to promote cancer burden by two combined events – “loss of brake and an acceleration”. Our results highlight the oncogenic role of the non-canonical base-pairing between miRNAs/mRNAs in uveal melanoma. MP41 cells were transfected with synthetic miRNA-16 vs miRNA-Control (66nM) (Dharmacon). Cells were harvested before transfection (0h) and then at 6h, 12h, 24h and 48h. RNA was extracted and the RIN was evaluated to ensure the good quality of RNA.

3号染色体短臂单体性（Chromosome 3p monosomy）与葡萄膜黑色素瘤（uveal melanoma）患者的不良临床预后密切相关。由于此类患者丢失了一份肿瘤抑制性微小RNA-16（miR-16）的拷贝，我们曾推测3p缺失会降低miR-16的表达量与活性，进而促进RNA去抑制与肿瘤负荷增加（即“刹车效应”丧失），这一现象在慢性淋巴细胞白血病（chronic lymphocytic leukemia）中已有报道。出乎意料的是，我们发现尽管存在3p单体性，miR-16的表达水平并未下降。与之相反，我们的结果显示葡萄膜黑色素瘤中miR-16的活性受到了损伤。本研究旨在解析RNA介导miR-16螯合的分子机制。通过对miR-16的RNA互作组（RNA interactome）进行分析，我们筛选得到两组基因集，提示miR-16存在两种截然不同的功能模式。除了CCND3、CDC25A等经典miR-16靶基因外，我们还鉴定出另一组与miR-16结合的RNA，我们将其命名为miR-16海绵吸附体（miR-16 sponges）。miR-16可与这些海绵吸附体RNA结合，但不会诱导其降解。从机制上来说，miR-16与RNA之间的非经典碱基配对（non-canonical base-pairing）可增强与癌细胞增殖相关的mRNA的稳定性。我们在葡萄膜黑色素瘤中验证了这一生物学效应：总生存期较差的患者，其miR-16海绵吸附体与经典miR-16靶基因的表达水平均更高。上述结果强烈提示，miR-16已无法再对其靶基因产生抑制作用，反而会促进致癌基因的RNA稳定性与蛋白质表达。利用我们建立的海绵特征（Sponge-signature）进行miR-16活性评估，其区分患者总生存期的效能可与当前基于拷贝数变异（copy number variations）与驱动突变（driver mutations）检测的方法相媲美。综上，由微小RNA（miRNA）螯合引发的miRNA功能缺失似乎通过两种协同事件促进肿瘤负荷增加——“丧失刹车功能”与“加速增殖”。我们的研究结果揭示了miRNA与mRNA之间的非经典碱基配对在葡萄膜黑色素瘤中的致癌作用。本实验中，我们将合成的miR-16与miR-对照分别转染MP41细胞（转染浓度为66nM，购自Dharmacon）；分别在转染前（0h）以及转染后6h、12h、24h、48h收集细胞。提取总RNA并检测RNA完整性数（RIN），以确保RNA质量合格。