重复转染 Aurora-A siRNA 对 SK-MES-1 或 A549 细胞纤毛生成和增殖的影响。

Effects of Repeated Aurora-A siRNA Transfection on Cilia Generation and Proliferation of SK-MES-1 or A549 Cells.

机构信息

1 Department of Respiratory Medicine, General Hospital of Command , Shenyang, China .

2 Department of Histology and Embryology, Shenyang Medical , Shenyang, China .

出版信息

Cancer Biother Radiopharm. 2018 Apr;33(3):110-117. doi: 10.1089/cbr.2017.2297.

DOI:10.1089/cbr.2017.2297

PMID:29641257

Abstract

BACKGROUND

Suppression of Aurora kinase A (Aurora-A, AURKA) by siRNA of Aurora-A (siAurora-A, siA) has been used in lung tumor treatment. However, the dose and frequency of gene transfection still need to be confirmed further. We imitated multiple administration of solid tumor and attempted to make out the effects of thrice transfection of siAurora-A on cilia generation and apoptosis of SK-MES-1 cells (SK) or A549 cells.

METHODS

The Aurora-A mRNA levels of cells cultured with serum for 6 d or without serum for 2, 4, or 6 d were examined with real-time quantitative PCR; Cells were transfected single or repeatedly with siAurora-A or siControl (siC), their Aurora-A mRNA levels were determined with PCR; Their cilia were examined with immunohistochemistry. Cell viability was measured with the MTT assay. Protein expression was analyzed with western blot.

RESULTS

Cell viability showed a downward trend along with the prolongation of starvation time to the second, fourth, and even to the sixth day in both types of cells. But, the expression level of Aurora-A mRNA flipped to rise at the sixth day instead of decreasing at the fourth day. Protein expression trend of total Aurora-A in the two groups was consistent with Aurora-A mRNA expression trend. Compared with siC-3 group (transfected three times with siControl), siAurora-A significantly reduced the Aurora-A mRNA expression in siA-3 group (transfected three times with siAurora-A). Similarly, the cell viability of siA-3 group was lower than that of siC-3 group. The cell viability of siC-3 group was higher than that of serum-free-6d group, but, levels of Aurora-A mRNA expression of siC-3 group had no difference with serum-free-6d group. Finally, among groups transfected once or three times or starved for 6 d, there was no significant difference of ciliated cell proportions in both types of cells respectively.

CONCLUSIONS

Repeated siAurora-A transfection decreased Aurora-A expression that resulted in effective suppression proliferation of SK-MES-1 or A549 cells, but did not affect cilia generation.

摘要

背景

利用 Aurora 激酶 A（Aurora-A，AURKA）的 siRNA（siAurora-A，siA）抑制已被用于肺癌肿瘤治疗。然而，基因转染的剂量和频率仍需要进一步确认。我们模拟了实体瘤的多次给药，并尝试确定 siAurora-A 三次转染对 SK-MES-1 细胞（SK）或 A549 细胞纤毛生成和细胞凋亡的影响。

方法

用实时定量 PCR 检测培养 6 天含血清或 2、4、6 天无血清的细胞中 Aurora-A mRNA 水平；细胞分别或重复用 siAurora-A 或 siControl（siC）转染，PCR 法检测 Aurora-A mRNA 水平；免疫组化法检测纤毛；MTT 法检测细胞活力；Western blot 法分析蛋白表达。

结果

两种细胞的细胞活力随饥饿时间的延长而呈下降趋势，从第二天到第四天甚至第六天都在下降。但是，Aurora-A mRNA 的表达水平在第六天反而上升，而不是在第四天下降。两组总 Aurora-A 的蛋白表达趋势与 Aurora-A mRNA 表达趋势一致。与 siC-3 组（转染 3 次 siControl）相比，siAurora-A 显著降低了 siA-3 组（转染 3 次 siAurora-A）的 Aurora-A mRNA 表达。同样，siA-3 组的细胞活力低于 siC-3 组。siC-3 组的细胞活力高于无血清 6d 组，但 siC-3 组的 Aurora-A mRNA 表达水平与无血清 6d 组无差异。最后，在单次或 3 次转染或饥饿 6d 的各组中，两种细胞的纤毛细胞比例均无显著差异。