RNA干扰转录因子激活蛋白4基因表达对结肠癌细胞株SW480的影响

目的观察转录因子激活蛋白4（AP-4）基因对结肠癌细胞株SW480生物学行为的影响。方法设计合成针对AP-4基因外显子7的小分子干扰RNA（siRNA）表达质粒,用脂质体转染SW480细胞,通过RT-PCR技术、Western印迹、四甲基偶氮唑盐实验（MTT法）、流式细胞仪和Transwell体外侵袭实验等检测该siRNA对SW480细胞基因表达、细胞增殖、细胞周期及细胞凋亡能力和浸润转移能力等生物学行为的影响。结果AP-4siRNA转染SW480细胞96h后,其AP-4mRNA水平下降了58%,培养液上清AP-4蛋白浓度下降了75%（P〈0.01）。细胞增殖受到明显抑制,抑制率达61%～78%;流式细胞仪检测结果显示,AP-4siRNA组SW480细胞凋亡率为（21.70±2.51）%,显著高于阴性对照组的（2.31±0.14）%（P〈0.01）,G0-G1期细胞比例增加（P〈0.01）,G2-M期细胞减少（P〈0.05）;Transwell体外侵袭实验显示,AP-4siRNA能明显抑制SW480细胞的体外侵袭力（P〈0.01）。结论应用siRNA技术沉默AP-4基因能有效抑制SW-480细胞AP-4的表达,进而抑制细胞的生长、增殖及诱导细胞的凋亡,为以AP-4为靶向的结肠癌基因治疗提供了新的思路和手段。

Inhibition of activator protein 4 gene expression by RNA interference in human colon carcinoma cell line SW480

OBJECTIVE: To construct an expression plasmid of siRNA against activator protein 4(AP-4) gene and to investigate its biological behavioral effect on human colon carcinoma cell line SW480. METHODS: The specific siRNA target exon 7 of AP-4 gene was constructed and transfected into SW480 cells by liposome. Expression levels of AP-4 mRNA and protein from SW480 after transfection were examined by RT-PCR and Western blot respectively. Cell proliferation was detected by cell counting and MTT assay. Flow cytometry(FCM) and Western blot were used to detect cell apoptosis and cell cycle. The invasiveness of SW480 cells in vitro was measured quantitatively by the matrigel invasion assay. RESULTS: After AP-4 siRNA transfection into SW480 cells for 96 hours, the cell AP-4 mRNA reduced by 57.8% and the AP-4 protein concentration in culture supernatant decreased by 75.2%. The cell growth was significantly inhibited by 61%-78%. The apoptosis rate of SW480 cells was significantly higher in AP-4 siRNA group than that in negative control group(21.7 +/- 2.51)% vs. (2.31 +/- 0.14)%, P<0.01]. Cells in G0-G1 phase increased by 22.43% and in G2-M phase decreased by 14.52% (P<0.05). AP-4 siRNA significantly inhibited AP-4-induced invasion of SW480 cells to matrigel (P<0.05). CONCLUSIONS: Silencing AP-4 gene by the siRNA technology can actively suppress the expression of AP-4 gene, and then inhibit the growth and proliferation of SW480 cell, and induce apoptosis of SW480 cell. The successful application of AP-4 siRNA extends the list of available therapeutic modalities in the treatment of human colon cancer.