Department of Experimental Oncology, Institute of Oncology Ljubljana, 1000 Ljubljana, Slovenia.
Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia.
Int J Mol Sci. 2021 Mar 17;22(6):3069. doi: 10.3390/ijms22063069.
Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.
通过特异性内皮细胞标志物靶向肿瘤血管代表了一种有前途的癌症治疗方法。在这里，我们的目的是构建一个不含抗生素抗性基因的质粒，该质粒编码短发夹 RNA (shRNA)，以同时靶向两个内皮细胞标志物 CD105 和 CD146，并通过基因电转移 (GET) 递送该质粒，结合照射 (IR) ，在体外测试其功能和治疗潜力。通过 qRT-PCR 测定靶向基因的沉默来评估质粒的功能。通过细胞毒性测定管形成测定和伤口愈合测定在小鼠内皮细胞 2H-11 中测定抗增殖和抗血管生成作用。还在恶性黑色素瘤肿瘤细胞系 B16F10 中评估了质粒构建体的功能。此外，通过 qRT-PCR 测量内皮细胞 2H-11 中 DNA 传感器 STING 和促炎细胞因子的诱导来测量潜在的免疫反应激活。我们证明了质粒构建是成功的，可以有效地沉默两个靶向基因的表达。由于沉默，在 2H-11 内皮细胞中证实了迁移率和血管生成潜力降低。此外，还确定了 DNA 传感器 STING 和促炎细胞因子的诱导，这可能会增加体内使用时的治疗效果。总之，我们成功构建了一种带有两个 shRNA 的新型质粒 DNA，这为进一步的体内试验提供了巨大的潜力。
