Di Martino Maria Teresa, Gullà Annamaria, Gallo Cantafio Maria Eugenia, Altomare Emanuela, Amodio Nicola, Leone Emanuela, Morelli Eugenio, Lio Santo Giovanni, Caracciolo Daniele, Rossi Marco, Frandsen Niels M, Tagliaferri Pierosandro, Tassone Pierfrancesco
Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy ; T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy.
Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy.
PLoS One. 2014 Feb 21;9(2):e89659. doi: 10.1371/journal.pone.0089659. eCollection 2014.
BACKGROUND & AIM: The miR-221/222 cluster is upregulated in malignant plasma cells from multiple myeloma (MM) patients harboring the t(4;14) translocation. We previously reported that silencing of miR-221/222 by an antisense oligonucleotide induces anti-MM activity and upregulates canonical miR-221/222 targets. The in vivo anti-tumor activity occurred when miR-221/222 inhibitors were delivered directly into MM xenografts. The aim of the present study was to evaluate the anti-MM activity of a novel phosphorothioate modified backbone 13-mer locked nucleic acid (LNA)-Inhibitor-miR-221 (LNA-i-miR-221) specifically designed for systemic delivery.
In vitro anti-MM activity of LNA-i-miR-221 was evaluated by cell proliferation and BrdU uptake assays. In vivo studies were performed with non-obese diabetic/severe combined immunodeficient (NOD.SCID) mice bearing t(4;14) MM xenografts, which were intraperitoneally or intravenously treated with naked LNA-i-miR-221. RNA extracts from retrieved tumors were analyzed for miR-221 levels and modulation of canonical targets expression. H&E staining and immunohistochemistry were performed on retrieved tumors and mouse vital organs.
In vitro, LNA-i-miR-221 exerted strong antagonistic activity against miR-221 and induced upregulation of the endogenous target p27Kip1. It had a marked anti-proliferative effect on t(4;14)-translocated MM cells but not on MM cells not carrying the translocation and not overexpressing miR-221. In vivo, systemic treatment with LNA-i-miR-221 triggered significant anti-tumor activity against t(4;14) MM xenografts; it also induced miR-221 downregulation, upregulated p27Kip1 and reduced Ki-67. No behavioral changes or organ-related toxicity were observed in mice as a consequence of treatments.
LNA-i-miR-221 is a highly stable, effective agent against t(4;14) MM cells, and is suitable for systemic use. These data provide the rationale for the clinical development of LNA-i-miR-221 for the treatment of MM.
在携带t(4;14)易位的多发性骨髓瘤（MM）患者的恶性浆细胞中，miR-221/222簇呈上调状态。我们之前报道过，反义寡核苷酸沉默miR-221/222可诱导抗MM活性并上调miR-221/222的经典靶标。当将miR-221/222抑制剂直接注射到MM异种移植瘤中时，可产生体内抗肿瘤活性。本研究的目的是评估一种专门设计用于全身给药的新型硫代磷酸酯修饰骨架13聚体锁核酸（LNA）-抑制剂-miR-221（LNA-i-miR-221）的抗MM活性。
通过细胞增殖和BrdU摄取试验评估LNA-i-miR-221的体外抗MM活性。对携带t(4;14) MM异种移植瘤的非肥胖糖尿病/严重联合免疫缺陷（NOD.SCID）小鼠进行体内研究，将裸LNA-i-miR-221经腹腔或静脉注射给药。对回收肿瘤的RNA提取物进行分析，检测miR-221水平以及经典靶标表达的调节情况。对回收的肿瘤和小鼠重要器官进行苏木精-伊红（H&E）染色和免疫组化分析。
在体外，LNA-i-miR-221对miR-221具有强大的拮抗活性，并诱导内源性靶标p27Kip1上调。它对携带t(4;14)易位的MM细胞具有显著的抗增殖作用，但对未携带该易位且未过表达miR-221的MM细胞没有作用。在体内，LNA-i-miR-221全身治疗引发了针对t(4;14) MM异种移植瘤的显著抗肿瘤活性；它还诱导miR-221下调，上调p27Kip1并降低Ki-67。治疗后未观察到小鼠出现行为变化或器官相关毒性。
LNA-i-miR-221是一种针对t(4;14) MM细胞的高度稳定、有效的药物，适用于全身应用。这些数据为LNA-i-miR-221用于MM治疗的临床开发提供了理论依据。
