Fathi Mojtaba, Yavari Kamal, Taghikhani Mohammad, Ghannadi Maragheh Mohammad
aDepartment of Clinical Biochemistry, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan bDepartment of Radiochemistry, Nuclear Sciences and Technology Research Institute (NSTRI) cDepartment of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Nucl Med Commun. 2015 Jun;36(6):636-45. doi: 10.1097/MNM.0000000000000292.
Serum and intracellular instability limits the therapeutic applications of short interfering RNA (siRNA) as a radiopharmaceutical. Chemical modifications like phosphorothioate (PS) substitution and 2'-O-methoxy (2'-O-Me) modifications could eliminate such limitations. In this study, the effects of PS and 2'-O-Me modifications at the backbone of siRNA on serum stability and RNA interference activity were investigated.
Fully PS and 2'-O-Me-modified type 1 insulin-like growth factor receptor (IGF-1R) siRNA was radiolabeled with lutetium-177 ((177)Lu) through p-SCN-Bn-DTPA as a chelator. After purification with Vivaspin and PD-10 columns, the radiolabs were examined for stability in serum by instant thin-layer chromatography and polyacrylamide gel electrophoresis. The level of IGF-1R in response to the modified and labeled IGF-1R siRNA was examined using RT-PCR and ELISA assay in colon cancer cells. The effects of such siRNA on the prevention of proliferation of colon cancer cells and its apoptosis were investigated using MTT assay and Annexin-V/propidium iodide double staining, respectively. Cellular accumulation quantities of the labeled and modified IGF-1R siRNA were determined using a γ-counter by taking advantage of (177)Lu as a γ-emitter.
Both the modified (177)Lu-siRNA complex and the modified nonlabeled siRNA showed significant stability in serum. The levels of IGF-1R mRNA and protein significantly decreased with both the labeled and nonlabeled IGF-1R siRNAs, but no such reduction in IGF-1R was observed with luciferase siRNA (P<0.01). Proliferation decreased significantly and apoptosis increased in the cells treated with modified (177)Lu-IGF-1R siRNA in comparison with either (177)Lu or labeled luciferase siRNA (P<0.001).
Uniform chemically modified siRNAs can form stable complexes with Lu that pronounce its cytotoxic effect through apoptosis in colon cancer cells.
血清和细胞内的不稳定性限制了短干扰RNA（siRNA）作为放射性药物的治疗应用。诸如硫代磷酸酯（PS）取代和2'-O-甲氧基（2'-O-Me）修饰等化学修饰可以消除此类限制。在本研究中，研究了siRNA主链上的PS和2'-O-Me修饰对血清稳定性和RNA干扰活性的影响。
通过作为螯合剂的对氨基苯硫酰基苄基二乙三胺五乙酸（p-SCN-Bn-DTPA），用镥-177（¹⁷⁷Lu）对完全PS和2'-O-Me修饰的1型胰岛素样生长因子受体（IGF-1R）siRNA进行放射性标记。用Vivaspin和PD-10柱纯化后，通过即时薄层色谱和聚丙烯酰胺凝胶电泳检查放射性标记物在血清中的稳定性。在结肠癌细胞中，使用逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）检测修饰并标记的IGF-1R siRNA作用下IGF-1R的水平。分别使用噻唑蓝（MTT）测定法和膜联蛋白V/碘化丙啶双染色法研究此类siRNA对结肠癌细胞增殖预防及其凋亡的影响。利用¹⁷⁷Lu作为γ发射体，通过γ计数器测定标记并修饰的IGF-1R siRNA的细胞摄取量。
修饰的¹⁷⁷Lu-siRNA复合物和修饰的未标记siRNA在血清中均表现出显著的稳定性。标记和未标记的IGF-1R siRNAs均使IGF-1R mRNA和蛋白水平显著降低，但荧光素酶siRNA未观察到IGF-1R的这种降低（P<0.01）。与¹⁷⁷Lu或标记的荧光素酶siRNA相比，用修饰的¹⁷⁷Lu-IGF-1R siRNA处理的细胞中增殖显著降低，凋亡增加（P<0.001）。
均匀化学修饰的siRNAs可与镥形成稳定的复合物，通过诱导结肠癌细胞凋亡发挥其细胞毒性作用。
