Nahar Smita, Singh Amrita, Morihiro Kunihiko, Moai Yoshihiro, Kodama Tetsuya, Obika Satoshi, Maiti Souvik
Academy of Scientific and Innovative Research (AcSIR) , Anusandhan Bhawan, 2 Rafi Marg, New Delhi 110001, India.
CSIR-Institute of Genomics and Integrative Biology , Mathura Road, Delhi 110025, India.
Biochemistry. 2016 Dec 20;55(50):7023-7032. doi: 10.1021/acs.biochem.6b00895. Epub 2016 Dec 5.
miRNAs constitute an important layer of gene regulation mediated by sequence-specific targeting of mRNAs. Aberrant expression of miRNAs contributes to a host of pathological states. Promoting cancer, miR-21 is upregulated in variety of cancers and promotes tumor progresion by suppressing a network of tumor suppressor genes. Here we describe a novel class of bicyclic RNA analogues, selenomethylene-locked nucleic acid (SeLNA), that display high affinity, improved metabolic stability, and increased potency for miR-21 inhibition. The thermal stability (T) for duplexes was increased significantly with incorporation of SeLNA monomers as compared to that of the unmodified DNA-RNA hybrid. A comprehensive thermodynamic profile obtained by isothermal titration calorimetry revealed a favorable increase in the enthalpy of hybridization for SeLNA containing DNA and target RNA heteroduplexes. SeLNA modifications displayed remarkable binding affinity for miR-21 target RNA with a K of ≤1.05 × 10 M. We also observed enhanced serum stability for SeLNA-RNA duplexes with a half-life of ≤36 h. These in vitro results were well correlated with the antisense activity in cancer cells imparting up to ∼91% inhibition of miR-21. The functional impact of SeLNA modifications on miR-21 inhibition was further gauged by investigating the migration and invasion characterisitics of cancer cells, which were drastically reduced to ∼49 and ∼55%, respectively, with SeLNA having four such modifications. Our findings demonstrate SeLNA as a promising candidate for therapeutics for disease-associated miRNAs.
微小RNA（miRNAs）构成了由mRNA的序列特异性靶向介导的基因调控的重要层面。miRNAs的异常表达会导致许多病理状态。促进癌症发生的miR-21在多种癌症中上调，并通过抑制一系列肿瘤抑制基因网络来促进肿瘤进展。在此，我们描述了一类新型的双环RNA类似物，即亚硒甲基锁定核酸（SeLNA），其对miR-21具有高亲和力、改善的代谢稳定性以及增强的抑制效力。与未修饰的DNA-RNA杂交体相比，掺入SeLNA单体后双链体的热稳定性（T m）显著提高。通过等温滴定量热法获得的全面热力学图谱显示，含SeLNA的DNA与靶RNA异源双链体的杂交焓有有利增加。SeLNA修饰对miR-21靶RNA表现出显著的结合亲和力，解离常数K d≤1.05×10⁻⁹ M。我们还观察到SeLNA-RNA双链体在血清中的稳定性增强，半衰期≤36小时。这些体外结果与癌细胞中的反义活性密切相关，对miR-21的抑制率高达约91%。通过研究癌细胞的迁移和侵袭特性，进一步评估了SeLNA修饰对miR-21抑制的功能影响，当SeLNA有四处此类修饰时，癌细胞的迁移和侵袭特性分别大幅降低至约49%和55%。我们的研究结果表明，SeLNA是与疾病相关的miRNAs治疗的有前景的候选物。
