Department of Nuclear Medicine, Kyungpook National University School of Medicine, Daegu, South Korea.
J Nucl Med. 2011 Nov;52(11):1756-63. doi: 10.2967/jnumed.111.090266. Epub 2011 Oct 12.
The purpose of this study was to investigate the enhanced therapeutic effect of the combined use of shRNA (small hairpin RNA) therapy for the hexokinase II (HKII) gene and (131)I human sodium iodide symporter (hNIS) as a gene therapy for in vitro and in vivo treatment of anaplastic thyroid carcinoma cells (ARO) in an animal model.
A recombinant lentivirus containing a plasmid with the hNIS gene driven by phosphoglycerate kinase promoter and green fluorescent protein (GFP) linked with an internal ribosome entry site sequence was produced. ARO cells were transfected with the virus and sorted by fluorescent activated cell sorting using GFP (ARO-NG). The messenger RNA expression of hNIS and GFP were evaluated with reverse-transcriptase polymerase chain reaction, and the function of hNIS was verified by (125)I uptake. The lentiviral vector expressing shRNA against HKII (Lenti-HKII shRNA) was constructed and used to infect ARO-NG cells. The effect of Lenti-HKII shRNA was evaluated by reverse-transcriptase polymerase chain reaction, (18)F-FDG uptake, and HK activity. An in vitro clonogenic assay was performed after Lenti-HKII shRNA therapy, (131)I therapy, and a combined therapy. The therapies were also applied in vivo to an animal model with an ARO-NG xenograft, and the effects were assessed with caliper measurements and (18)F-FDG PET.
ARO-NG cells showed an (125)I uptake 76-fold higher than the parent ARO cells. Compared with the uninfected ARO-NG cells, ARO-NG cells infected with Lenti-HKII shRNA had lower HKII messenger RNA expression, lower (18)F-FDG uptake, and HK activity. The proliferation of ARO-NG cells was inhibited by (131)I and Lenti-HKII shRNA therapies and further inhibited by the combined (131)I and Lenti-HKII shRNA therapy. Both the Lenti-HKII shRNA therapy and the (131)I therapy inhibited in vivo tumor growth in the tumor xenograft model. The combined Lenti-HKII shRNA and (131)I therapy resulted in a further decrease of tumor growth.
Our results suggest that the combined HKII shRNA and (131)I therapy has a stronger antitumor effect than either the (131)I therapy or the HKII shRNA alone. Therefore, this combined therapy could be used as a powerful strategy for treating anaplastic thyroid carcinoma.
本研究旨在探讨短发夹 RNA (shRNA) 疗法联合应用己糖激酶 II (HKII) 基因和人钠碘转运体 (hNIS) 治疗甲状腺未分化癌 (ATC) 细胞的体外和体内治疗效果，构建动物模型。
采用磷酸甘油酸激酶启动子驱动的 hNIS 基因和绿色荧光蛋白 (GFP) 连接内部核糖体进入位点序列的重组慢病毒载体。通过荧光激活细胞分选技术，使用 GFP 对 ARO 细胞进行病毒转染和分选 (ARO-NG)。采用逆转录聚合酶链反应 (RT-PCR) 检测 hNIS 和 GFP 的信使 RNA 表达，通过 125I 摄取验证 hNIS 的功能。构建表达 HKII shRNA 的慢病毒载体 (Lenti-HKII shRNA)，并用于感染 ARO-NG 细胞。通过 RT-PCR、18F-FDG 摄取和 HK 活性评估 Lenti-HKII shRNA 的作用。在 Lenti-HKII shRNA 治疗、131I 治疗和联合治疗后进行体外集落形成实验。还将这些治疗方法应用于 ARO-NG 异种移植的动物模型中，通过卡尺测量和 18F-FDG PET 评估疗效。
ARO-NG 细胞的 125I 摄取量比亲本 ARO 细胞高 76 倍。与未感染的 ARO-NG 细胞相比，感染 Lenti-HKII shRNA 的 ARO-NG 细胞的 HKII 信使 RNA 表达降低，18F-FDG 摄取和 HK 活性降低。131I 和 Lenti-HKII shRNA 治疗均可抑制 ARO-NG 细胞的增殖，联合治疗可进一步抑制。Lenti-HKII shRNA 治疗和 131I 治疗均可抑制肿瘤异种移植模型中的肿瘤生长，联合 Lenti-HKII shRNA 和 131I 治疗可进一步降低肿瘤生长。
本研究结果表明，与单独使用 131I 治疗或 HKII shRNA 治疗相比，联合 HKII shRNA 和 131I 治疗具有更强的抗肿瘤作用。因此，这种联合治疗可能成为治疗甲状腺未分化癌的有效策略。
