Department of Molecular Virology, Pasteur Institute of Iran, Tehran 1316943551, Iran.
Department of Microbiology, School of Biology, College of Science, University of Tehran, 1417614411, Iran.
Iran Biomed J. 2023 Nov 1;27(6):349-56. doi: 10.61186/ibj.3963.
The E6 oncoprotein of HPV plays a crucial role in promoting cell proliferation and inhibiting apoptosis, leading to tumor growth. Non-viral vectors such as nona-arginine (R9) peptides have shown to be potential as carriers for therapeutic molecules. This study aimed to investigate the efficacy of nona-arginine in delivering E6 shRNA and suppressing the E6 gene of HeLa cells in vitro.
HeLa cells carrying E6 gene were treated with a complex of nona-arginine and E6 shRNA. The complex was evaluated using gel retardation assay and FESEM microscopy. The optimal N/P ratio for R9 peptide to transfect HeLa cells with luciferase gene was determined. Relative real-time PCR was used to evaluate the efficiency of mRNA suppression efficiency for E6 shRNA, while the effect of E6 shRNA on cell viability was measured using an MTT assay.
The results indicated that R9 efficiently binds to shRNA and effectively transfects E6 shRNA complexes at N/P ratios greater than 30. Transfection with R9 and PEI complexes resulted in a significant toxicity compared to the scrambled plasmid, indicating selective toxicity for HeLa cells. Real-time PCR confirmed the reduction of E6 mRNA expression levels in the cells transfected with anti-E6 shRNA.
The study suggests that R9 is a promising non-viral gene carrier for transfecting E6 shRNA in vitro, with significant transfection efficiency and minimal toxicity.
HPV 的 E6 癌蛋白在促进细胞增殖和抑制细胞凋亡方面发挥着关键作用，导致肿瘤生长。非病毒载体，如九聚精氨酸（R9）肽，已被证明是治疗分子载体的潜在选择。本研究旨在研究九聚精氨酸在递送 E6 shRNA 和体外抑制 HeLa 细胞中 E6 基因方面的功效。
用携带 E6 基因的 HeLa 细胞处理非病毒载体 R9 肽和 E6 shRNA 的复合物。通过凝胶阻滞试验和 FESEM 显微镜评估复合物。确定 R9 肽转染 HeLa 细胞携带荧光素酶基因的最佳 N/P 比。使用相对实时 PCR 评估 E6 shRNA 的 mRNA 抑制效率，同时使用 MTT 测定 E6 shRNA 对细胞活力的影响。
结果表明，R9 能够有效地结合 shRNA，并在 N/P 比大于 30 时有效地转染 E6 shRNA 复合物。与对照质粒相比，R9 和 PEI 复合物的转染导致明显的毒性，表明对 HeLa 细胞具有选择性毒性。实时 PCR 证实了转染抗 E6 shRNA 的细胞中 E6 mRNA 表达水平的降低。
该研究表明，R9 是一种很有前途的非病毒基因载体，可用于体外转染 E6 shRNA，具有显著的转染效率和最小的毒性。
