Zhang Yan, Wu Haibo, Zhang Man, Jiang Yali, Zhuo Weiwei, Zhang Yong, Hua Song
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province, 712100, People's Republic of China,
Tumour Biol. 2015 Jun;36(6):4339-48. doi: 10.1007/s13277-015-3073-3. Epub 2015 Jan 17.
The aim of this study was to decide whether nicotinamide (NA) could induce apoptosis of F9 mouse teratocarcinoma stem cells (MF9) by downregulation of special AT-rich sequence binding protein 1 (SATB1) expression. We used different concentrations of NA (0, 1.5, 2, and 2.5 mmol/L) to treat MF9 cells and analyze SATB1 expression by RT-qPCR and Western blotting; in addition, the cell proliferation was detected in a microplate reader with Cell Counting Kit-8 (CCK-8), and the cell cycle and apoptosis were analyzed using flow cytometry. We found that the expression of SATB1 was decreased significantly in NA-treated groups than in the control group, and its expression level was inversely related to the NA concentration. In addition, CCK-8 analysis showed that NA significantly inhibited the proliferation of MF9 cells, and flow cytometry showed that NA blocked MF9 cells to G1 phase and significantly promoted apoptosis in any treated groups. To confirm the results, we constructed small interference RNA (siRNA) targeting at mouse SATB1 and transferred into MF9 cells. The results indicated that the expression of SATB1 in both mRNA and protein levels was significantly decreased after cells transferred with siRNA sequence for 48 h, the proliferation of MF9 cells was significantly inhibited, and most of MF9 cells were blocked at G1 phase, and the apoptosis rate was increased obviously. The results showed that NA could inhibit the proliferation and induce apoptosis of MF9 cells. These findings might be used as an efficient candidate for teratocarcinoma therapy.
本研究的目的是确定烟酰胺（NA）是否可以通过下调富含AT序列的特异性结合蛋白1（SATB1）的表达来诱导F9小鼠畸胎瘤干细胞（MF9）凋亡。我们使用不同浓度的NA（0、1.5、2和2.5 mmol/L）处理MF9细胞，并通过RT-qPCR和蛋白质印迹法分析SATB1的表达；此外，使用细胞计数试剂盒-8（CCK-8）在酶标仪中检测细胞增殖，并使用流式细胞术分析细胞周期和凋亡情况。我们发现，与对照组相比，NA处理组中SATB1的表达显著降低，且其表达水平与NA浓度呈负相关。此外，CCK-8分析表明，NA显著抑制MF9细胞的增殖，流式细胞术显示，NA将MF9细胞阻滞在G1期，并在任何处理组中均显著促进凋亡。为了证实结果，我们构建了靶向小鼠SATB1的小干扰RNA（siRNA）并转入MF9细胞。结果表明，用siRNA序列转染细胞48小时后，SATB1的mRNA和蛋白水平表达均显著降低，MF9细胞的增殖受到显著抑制，大多数MF9细胞被阻滞在G1期，凋亡率明显增加。结果表明，NA可以抑制MF9细胞的增殖并诱导其凋亡。这些发现可能成为畸胎瘤治疗的有效候选方法。
