Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine, Ministry of Education, Chongqing Medical University, Chongqing 400016, PR China.
Oncol Rep. 2012 Oct;28(4):1362-8. doi: 10.3892/or.2012.1911. Epub 2012 Jul 13.
PR39, a porcine cathelicidin rich in the amino acids proline and arginine can interact with the negatively charged component of the cell surface, and rapidly penetrate cell membranes. Therefore, we hypothesized that PR39, as a membrane penetrating peptide (MPP), could be exploited as a novel carrier to deliver siRNA into the cell cytoplasm in order to knockdown target gene expression. Firstly, a complex formation of PR39 with siRNA and its cellular colocalization were investigated in our studies. Further, we optimized the ratio of the PR39/siRNA complex, cell/complex incubation period and the concentration of siRNA. The results suggest that PR39 could form a complex with siRNA, and mediate translocation of the siRNA into 4T1 cells. The optimal ratio of siRNA with PR39 was 1:90 which was found to have a maximum Stat3 gene silencing effect after 48 h treatment. Moreover, 4T1 cell proliferation, cell cycle, invasion and migration were investigated. The results suggested that Stat3 knockdown could not result in 4T1 cell proliferation inhibition and cell cycle arrest, while invasion and migration of 4T1 cells were strongly inhibited. Notably, the data also showed that in addition to inhibition of carcinogenesis, single PR39 may play a role in cell invasion and migration. PR39 and Stat3 siRNA displayed synergistic biological effects in inhibiting cell invasion and migration of 4T1 cells, which was more prominent compared with the popular Lipofectamine delivery system.
富含脯氨酸和精氨酸的猪 cathelicidin（PR39）可以与细胞表面的带负电荷的成分相互作用，并迅速穿透细胞膜。因此，我们假设 PR39 可以作为一种新型载体，利用其穿透细胞膜的肽（MPP）特性将 siRNA 递送至细胞质中，以达到敲低靶基因表达的目的。首先，我们研究了 PR39 与 siRNA 的复合物形成及其在细胞内的共定位。进一步，我们优化了 PR39/siRNA 复合物的比例、细胞/复合物孵育时间和 siRNA 的浓度。结果表明，PR39 可以与 siRNA 形成复合物，并介导 siRNA 转染至 4T1 细胞。我们发现，PR39 与 siRNA 的最佳比例为 1:90，在 48 小时处理后可达到最大的 Stat3 基因沉默效果。此外，我们还研究了 4T1 细胞的增殖、细胞周期、侵袭和迁移。结果表明，Stat3 基因沉默不会导致 4T1 细胞增殖抑制和细胞周期停滞，但可强烈抑制 4T1 细胞的侵袭和迁移。值得注意的是，这些数据还表明，除了抑制肿瘤发生外，单一 PR39 可能在细胞侵袭和迁移中发挥作用。PR39 和 Stat3 siRNA 在抑制 4T1 细胞的侵袭和迁移方面表现出协同的生物学效应，与流行的 Lipofectamine 递送系统相比，这种协同效应更加显著。
