莱菔硫烷诱导急性髓系白血病KG1a和KG1细胞G_(2)/M期阻滞的作用和相关机制

Effect and Mechanism of Sulforaphane on G_(2)/M Phase Arrest of Acute Myeloid Leukemia KG1a and KG1 Cells

目的:研究莱菔硫烷(sulforaphane,SFN)对急性髓系白血病细胞G_(2)/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G_(2)/M期细胞明显增多,当SFN浓度为8μmol/L时,KG1a细胞在G_(2)/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8μmol/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12μmol/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),SFN8和12μmol/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G_(2)/M期。

Objective: To investigate the effect of sulforaphane( SFN) on G_(2)/M phase arrest of acute myeloid leukemia cells and its molecular mechanism. Methods: KG1 a and KG1 cells 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 KG1 a 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 G_(2)/M phase were increased from 11. 9% to 54. 0% in KG1 a cells and 18. 5% to83. 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 KG1 a cells after treated by SFN. The heat-map graph showed that SFN could change the relevant genes of the cell cycle in KG1 a cells. After SFN treatment,the mRNA level of P53 and P21 were significantly increased in KG1 and KG1 a 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 kG1 a 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 μmol/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 G_(2)/M phase by activating P53 signaling pathway,which can inhibit the expression of CDC2 and the activity of CDC2/cyclinB1.