【摘要】 目的调节蛋白混合性白血病(regulatory protein mixed lineage leukaemia,MLL)是常见的复发/难治性急性白血病,目前仍无标准有效的治疗方案,急需研发新的靶向药物。本研究旨在检测小干扰RNA(siRNA)沉默人类组蛋白赖氨酸甲基转移酶hDOT1L基因表达对人单核细胞白血病细胞株THP-1细胞增殖的影响,探讨hDOT1L基因在白血病发病中的作用机制。方法利用LipofectamineTM2000将hDOT1LsiRNA转染体外培养的THP-1细胞,用四甲基偶氮唑蓝(MTT)法检测hDOT1LsiRNA单独或与地西他滨(Decitabine)联合应用对THP-1细胞增殖的影响。采用RT-PCR法检测THP-1细胞hDOT1L、HOXA9、HOXA10和MLL-AF10(MLLT10)mRNA表达水平,蛋白质印迹法检测THP-1细胞H3K79甲基化水平、hDOT1L蛋白表达水平及PI3K/AKT通路中关键分子表达变化。结果 MTT结果显示,与对照组相比(0.17±0.02),hDOT1LsiRNA单独(0.56±0.04)或与地西他滨联合应用(0.66±0.06)48h后可明显抑制THP-1细胞增殖,F=284.225,P<0.001。RT-PCR结果表明,THP-1细胞的HOXA9、HOXA10、MLLT10和hDOT1LmRNA表达水平在hDOT1LsiRNA组分别为0.34±0.06、0.27±0.06、0.32±0.08和0.26±0.07;在hDOT1LsiRNA+Decitabine组分别为0.15±0.06、0.14±0.05、0.16±0.06和0.13±0.06;与对照组(0.92±0.18、0.77±0.13、0.79±0.13和0.68±0.14)比较明显下降,差异有统计学意义,均P<0.001。蛋白质印迹法检测monoH3K79、di-H3K79、tri-H3K79蛋白表达水平在对照组分别为0.54±0.06、0.58±0.09和0.75±0.05,hDOT1LsiRNA组分别为0.44±0.02、0.31±0.06、0.41±0.09,hDOT1LsiRNA+Decitabine组分别为0.38±0.08、0.21±0.03、0.27±0.04,均较对照组明显降低,均P<0.001;Control siRNA组细胞内T-AKT、p-AKT、PI3K及hDOT1L蛋白表达水平分别为0.70±0.15、0.71±0.13、0.49±0.14、0.72±0.05,hDOT1LsiRNA组分别为0.64±0.12、0.51±0.04、0.30±0.04和0.36±0.04,hDOT1LsiRNA+Decitabine组分别为0.65±0.05、0.31±0.09、0.22±0.04和0.24±0.04,后两组与Control siRNA组相比,细胞内p-AKT、PI3K及hDOT1L蛋白表达水平明显下降,差异均有统计学意义,P<0.001;而总AKT(T-AKT)蛋白表达水平在各组间比较差异均无统计学意义,F=0.667,P=0.578。结论 hDOT1L基因可能通过增加白血病细胞H3K79基因甲基化水平,诱导MLL相关基因及PI3K/AKT通路中关键分子的表达,促进白血病细胞增殖而参与白血病发病机制。hDOT1L有望成为治疗白血病的新靶标,hDOT1L抑制剂与地西他滨联合应用有望进一步提高白血病治疗疗效。更多还原

【Abstract】 OBJECTIVE The purpose of this study was to investigate the possible mechanism of hDOT1 Lin the pathogenesis of leukemia.We explored the effect of a small interfering RNA(siRNA),which could silence a new human histone lysine methyltransferase hDOT1 Lgene,on the proliferation of human monocytic leukemia cell line THP-1cells.We also explored the possible mechanism by detecting the methylation level of H3K79 and the expression levels of key molecules.METHODS The hDOT1 LsiRNA chemically synthesized was transfected into THP-1cells with lipofectamineTM2000 in vitro.The proliferation of THP-1cells transfected with hDOT1 LsiRNA was detected by MTT.Trypan blue staining was used to detect the survival rate change of THP-1cells transfected with hDOT1 LsiRNA.Real time-PCR method was used to detect the expression levels of HOXA9,HOXA10,MLLT10,and hDOT1 L mRNA.Western Blot was used to detect the expression levels of H3K79 and the key molecules in PI3K/AKT pathway.We analyzed the effects of hDOT1 LsiRNA alone or in combination with decitabine on the expression levels of these molecules.RESULTS The results by MTT showed that the proliferation of THP-1cells was inhibited significantly(F=284.225,P<0.001)after48hlater in hDOT1 LsiRNA group(0.56±0.04)and hDOT1LsiRNA+Decitabine group(0.17±0.02),compared with the control group(0.66±0.06).The expression levels of HOXA9,HOXA10,MLLT10 and hDOT1L mRNA decreased by RT-PCR in THP-1cells were 0.34±0.06,0.27±0.06,0.32±0.08 and 0.26±0.07 in hDOT1LsiRNA group;0.15±0.06,0.14±0.05,0.16±0.06,0.13± 0.06 in hDOT1LsiRNA+Decitabine group;0.92±0.18,0.77±0.13,0.79±0.13,0.68±0.14 in control group.They were statistical significance(Fvalues were 92.439,93.352,99.023,63.314,all P<0.001).The expression levels of mono-H3K79,di-H3K79 and tri-H3K79 proteins decreased by Western Blot method were 0.54±0.06,0.58±0.09,0.75±0.05 in the control group,0.44±0.02,0.31±0.06,0.41±0.09 in hDOT1LsiRNA group,0.38±0.08,0.21±0.03,0.27±0.04 in hDOT1LsiRNA+Decitabine group respectively.Compared with the control group,the expression levels of the above proteins in the hDOT1 LsiRNA group and the hDOT1 L siRNA+Decitabine group were statistically significant(P<0.001).The protein expression levels of T-AKT,p-AKT,PI3 Kand hDOT1Lin Control siRNA group,Decitabine group,hDOT1 LsiRNA group,and hDOT1LsiRNA+Decitabine group were(0.70±0.15,0.71±0.13,0.49±0.14,0.72±0.05),(0.68±0.06,0.53±0.03,0.36±0.08,0.51±0.10),(0.64±0.12,0.51±0.04,0.30±0.04,0.36±0.04)and(0.65±0.05,0.31±0.09,0.22±0.04,0.24±0.04)respectively.Compared with Control siRNA group,the protein expression levels ofp-AKT,PI3 Kand hDOT1Lin Decitabine group,hDOT1 LsiRNA group,and hDOT1LsiRNA+Decitabine group were significantly decreased,and it had statistical significance(Fvalues 38.099,16.450,102.302,all P<0.001);the protein expression level of T-AKT had no statistical significance among each group(P>0.05).CONCLUSIONS hDOT1 Lgene may be involved in the pathogenesis of leukemia by increasing the methylation level of H3K79 in leukemia cells,and inducing the expression of key molecules in MLL related genes and PI3K/AKT pathways,and promoting the proliferation of leukemic cells.hDOT1 Lis expected to be a new target for leukemia,and the combination of hDOT1 Linhibitors and decitabine can improve the therapeutic efficacy of leukemia.更多还原