Objective: To investigate the effect of sulforaphane (SFN) on G2/M phase arrest of acute myeloid leukemia cells and its molecular mechanism.
Methods: KG1a and KG1cells were treated by different concentrations of SFN for 48 h. Flow cytometry (FCM) was used to analyze the phase distribution of cell cycle. High-throughput sequencing was used to detect the effect of SFN on the expression of cell cycle related genes in KG1a cells. The mRNA expression of P53, P21, CDC2 and CyclinB1 were detected by qPCR. The protein expression of P53, CDC2, P-CDC2 and CyclinB1 were detected by Western blot.
Results: Cells in the G2/M phase were increased from 11.9% to 54.0% in KG1a cells and 18.5% to 83.3% in KG1 cells after treated by SFN (8 μ mol / L) for 48 hours（P<0.001）. KEGG analysis indicated that P53 pathway was enriched in KG1a cells after treated by SFN. The heat-map graph showed that SFN could change the relevant genes of the cell cycle in KG1a cells. After SFN treatment, the mRNA level of P53 and P21 were significantly increased in KG1 and KG1a cells（P<0.05 or P<0.01）. The mRNA level of CDC2 showed a decrease trend with the increasing dose of SFN. At the dosage of 8 μmol /L, the mRNA expression levels of CDC2 was significantly lower than that in control group（P<0.05）. At the same time, the protein level of P53 was significantly increased in KG1 and kG1a cells after treated by SFN（P<0.05）. The protein level of CDC2 showed a decrease trend with the increasing dose of SFN in a dose manner（r=0.9482 and r=0.8977）. The protein levels of CDC2 in SFN 8 and 12 μ mol/L groups were significantly lower than that in control group(P<0.05, P<0.01). The protein levels of P-CDC2 was increased. But the change of mRNA and protein level of CyclinB1 was not significant.
Conclusion: SFN induces leukemia cells to block in G2/M phase by activating P53 signaling pathway, which can inhibit the expression of CDC2 and the activity of CDC2/cyclinB1.
题目: 莱菔硫烷诱导急性髓系白血病KG1a和KG1细胞G2/M期阻滞的作用和相关机制.
目的: 研究莱菔硫烷（sulforaphane, SFN）对急性髓系白血病细胞G2/M期阻滞的作用和相关机制.
方法: 不同浓度的SFN作用KG1a和KG1细胞48 h后，流式细胞术检测细胞周期的变化, 利用高通量转录组测序技术检测SFN对KG1a细胞周期相关基因表达情况的影响；采用实时荧光定量PCR（qPCR）检测P53、P21、CDC2、CyclinB1的mRNA表达。采用Western blot检测 P53、CDC2、P-CDC2和CyclinB1的蛋白表达.
结果: 经SFN作用48 h后，KG1a和KG1细胞的G2/M期细胞明显增多，当SFN浓度为8 μmol/L时，KG1a细胞在G2/M期从11.9%上升至54.0%，KG1细胞从18.5%上升至83.3%（P<0.001）。SFN作用KG1a细胞后，KEGG通路分析结果显示，差异表达基因显著富集P53信号通路。热图结果显示，P53和P21基因表达上调，CDC2基因表达下调。KG1a和KG1细胞经SFN作用后，p53和P21的mRNA水平均出现上调（P<0.05或P<0.01）。随着SFN剂量的上升，CDC2的mRNA水平呈下调趋势，当SFN为8 μmol/L时，CDC2的mRNA表达水平显著低于对照组（P<0.05）。经SFN作用KG1a和KG1后，p53的蛋白水平出现上调，SFN 8和12 μmol/L浓度组均高于对照组，差异有统计学意义（P<0.05）。两组细胞经SFN作用后CDC2的蛋白水平均明显下降，且呈浓度依赖趋势(r=0.9482和 r=0.8977)，SFN 8和12 μmol/L浓度组CDC2蛋白水平均明显低于对照组（P<0.05或P<0.01），P-CDC2蛋白表达水平上调。CyclinB1的蛋白和mRNA表达水平一致，变化不明显.
结论: SFN可能通过激活P53信号通路，进一步抑制CDC2表达和CDC2/CyclinB1复合物的活性，诱导急性髓系白血病KG1a和KG1细胞阻滞在G2/M期.