Abnormal activation of the IGF1R signaling pathway accelerates melanoma development and metastases. RNAi systems with complex cloning procedures and unsatisfactory efficiency in suppressing gene expression have become the technical difficulties that hinder their utility when studying gene knockdown. Here we established a simplified adenovirus-mediated gene knockdown system by which a single adenoviral vector carries multiple siRNA fragments that can effectively suppress IGF1R expression in melanoma cells. We first generated the adenovirus that simultaneously expresses three human or mouse siRNAs targeting IGF1R (AdRIGF1R-OK). qRT-PCR and immunofluorescence staining revealed that IGF1R expression was significantly decreased in the melanoma cells that were infected with AdRIGF1R-OK. Bioluminescence imaging showed that the size of the tumor formed by the xenografts infected with AdRIGF1R-OK was significantly smaller than that of the controls. Annexin V-FITC flow cytometry assay, immunofluorescence staining for cleaved caspase-3, and Hoechst staining showed that more cells underwent apoptosis after infection with AdRIGF1R-OK. Luciferase reporter assay, crystal violet cell viability assay, and cell-cycle analysis showed that the proliferation of melanoma cells infected with AdRIGF1R-OK was significantly decreased compared to the controls. This study demonstrates that the OK system is effective in silencing gene expression, with promising potential to treat melanoma and other diseases.

IGF1R信号通路的异常激活加速了黑色素瘤的发展和转移。RNAi系统具有复杂的克隆程序，并且在抑制基因表达方面效率不高，已经成为阻碍研究基因敲除的实用性的技术难题。在这里，我们建立了一个简化的腺病毒介导的基因敲低系统，通过该系统，单个腺病毒载体携带多个可有效抑制黑素瘤细胞中IGF1R表达的siRNA片段。我们首先生成了腺病毒，该腺病毒同时表达靶向IGF1R的三种人或小鼠siRNA（AdRIGF1R-OK）。qRT-PCR和免疫荧光染色显示，在被AdRIGF1R-OK感染的黑色素瘤细胞中，IGF1R表达显着降低。生物发光成像显示，被AdRIGF1R-OK感染的异种移植物形成的肿瘤大小显着小于对照。Annexin V-FITC流式细胞术测定，裂解的caspase-3的免疫荧光染色和Hoechst染色显示，AdRIGF1R-OK感染后更多的细胞发生凋亡。萤光素酶报告基因分析，结晶紫细胞活力分析和细胞周期分析表明，与对照组相比，AdRIGF1R-OK感染的黑色素瘤细胞的增殖明显降低。这项研究表明，OK系统可有效沉默基因表达，具有治疗黑素瘤和其他疾病的潜力。裂解caspase-3的免疫荧光染色和Hoechst染色显示，AdRIGF1R-OK感染后更多的细胞发生凋亡。萤光素酶报告基因分析，结晶紫细胞活力分析和细胞周期分析表明，与对照组相比，AdRIGF1R-OK感染的黑色素瘤细胞的增殖明显降低。这项研究表明，OK系统可有效沉默基因表达，具有治疗黑素瘤和其他疾病的潜力。裂解caspase-3的免疫荧光染色和Hoechst染色显示，AdRIGF1R-OK感染后更多的细胞发生凋亡。萤光素酶报告基因分析，结晶紫细胞活力分析和细胞周期分析表明，与对照组相比，AdRIGF1R-OK感染的黑色素瘤细胞的增殖明显降低。这项研究表明，OK系统可有效沉默基因表达，具有治疗黑素瘤和其他疾病的潜力。细胞周期分析表明，与对照组相比，感染了AdRIGF1R-OK的黑色素瘤细胞的增殖明显减少。这项研究表明，OK系统可有效沉默基因表达，具有治疗黑素瘤和其他疾病的潜力。细胞周期分析表明，与对照组相比，感染了AdRIGF1R-OK的黑色素瘤细胞的增殖明显减少。这项研究表明，OK系统可有效沉默基因表达，具有治疗黑素瘤和其他疾病的潜力。