Rodríguez-Vicente Ana E, Quwaider Dalia, Benito Rocío, Misiewicz-Krzeminska Irena, Hernández-Sánchez María, de Coca Alfonso García, Fisac Rosa, Alonso José-María, Zato Carolina, de Paz Juan Francisco, García Juan Luis, Sarasquete Ma Eugenia, Hernández José Ángel, Corchado Juan M, González Marcos, Gutiérrez Norma C, Hernández-Rivas Jesús-María
Servicio de Hematología, IBSAL, IBMCC, CIC, Universidad de Salamanca, CSIC, Hospital Universitario, Salamanca, Spain.
National Medicines Institute, Warsaw, Poland.
BMC Cancer. 2015 Apr 8;15:238. doi: 10.1186/s12885-015-1212-2.
MicroRNAs are known to inhibit gene expression by binding to the 3'UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients.
By applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3'UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status, HSP90B1 expression and clinico-biological data were assessed.
HSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome.
HSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target.
已知微小RNA通过与靶转录本的3'非翻译区（3'UTR）结合来抑制基因表达。最近有报道称，miR-223的下调在慢性淋巴细胞白血病（CLL）中具有预后意义。然而，尚无证据表明该微小RNA在CLL患者中的致病机制。
通过应用下一代测序技术，我们在24%的CLL患者中检测到一种常见的多态性（rs2307842），该多态性破坏了HSP90B1 3'UTR中miR-223的结合位点。我们通过荧光素酶测定和miR-223的异位表达研究了miR-223是否直接靶向HSP90B1。采用定量实时聚合酶链反应和蛋白质印迹法测定CLL患者中HSP90B1的表达。评估了rs2307842状态、HSP90B1表达与临床生物学数据之间的关系。
HSP90B1通过与假定的miR-223结合位点相互作用，是miR-223的直接靶点。配对样本（CD19+细胞组分和非CD19+细胞组分）分析显示，rs2307842和未突变的IGHV基因的存在决定了CLL患者B淋巴细胞中HSP90B1的过表达。蛋白质印迹法在蛋白质水平证实了这些结果。值得注意的是，HSP90B1过表达独立预测CLL患者首次治疗的时间较短。相比之下，rs2307842的存在与预后无关。
HSP90B1是miR-223的直接靶基因。我们的结果为携带miR-223下调的CLL患者预后不良的原因提供了一个合理的解释，指出HSP90B1是CLL中的一种新的致病机制和一个有前景的治疗靶点。
