DOT1L对骨肉瘤MG63细胞生物学行为的影响及其作用机制

Effect of DOT1L on biological behavior of osteosarcoma MG-63 cell and its mechanism

目的探讨抑制组蛋白H3赖氨酸79(H3K79)甲基转移酶DOT1L基因对骨肉瘤MG63细胞增殖、迁移、DNA损伤的影响及可能的作用机制。方法将hDOT1L-shRNA质粒转染至骨肉瘤MG63细胞(hDOT1L-shRNA组),同时设置shRNA阴性对照组(Scramble组)。采用实时荧光定量PCR检测DOT1L mRNA的表达水平,MTT法检测细胞增殖能力,划痕实验检测细胞迁移能力,TUNEL法和彗星实验检测细胞DNA损伤。Western blot检测MG63细胞中DOT1L、H3K79me2、Histone H3、Sirt1、XPA蛋白的表达水平。结果转染后,与Scramble组相比,hDOT1L-shRNA组MG63细胞中DOT1L mRNA及蛋白表达水平下调(P<0.05),细胞增殖能力降低(P<0.05),细胞迁移率降低(P<0.05),DNA损伤程度加剧(P<0.05)。Western blot检测结果显示,与Scramble组相比,hDOT1L-shRNA组MG63细胞中细胞Histone H3总量差异无统计学意义(P>0.05),其他DOT1L、H3K79me2、Sirt1、XPA蛋白表达水平均降低。结论DOT1L可能通过DOT1L/H3K79me2-SIRT1-XPA信号通路抑制骨肉瘤MG63细胞增殖、迁移并诱导其DNA损伤。

Objective To investigate the effect of histone H3 K79 methyltransferase disruptor of telomeric silencing-1 Like(DOT1 L)gene on the proliferation,apoptosis and migration of osteosarcoma MG63 cells and its possible mechanism.Methods hDOT1 L-shRNA was transfected into MG63 cells(hDOT1 L-shRNA),while shRNA negative control group(Scramble group)was set up.The expression of DOT1 L was detected by the real-time quantitative PCR,the ability of cell proliferation was detected by MTT method,the ability of cell migration was detected by scratch test,and the DNA damage was detected by TUNEL and Comet assay.The expression of DOT1 L,H3 K79 me2,Histone H3,Sirt1 and XPA proteins in UMG63 cells were detected by Western blot.Results Compared with the Scramble group,the DOT1 L mRNA and protein levels of MG63 cells in the hDOT1 L-shRNA group were significantly down-regulated(P<0.05);and the cell proliferation ability was significantly reduced(P<0.05),the cell migration ability was significantly reduced(P<0.05),and the DNA damage was significantly increased(P<0.05).Western blot showed that compared with the Scramble group,the protein expression levels of DOT1 L,H3 K79 me2,Sirt1 and XPA in the hDOT1 L-shRNA group were significantly reduced(P<0.05).Conclusion DOT1 L may inhibit the proliferation and migration of osteosarcoma MG63 cells and promote DNA damage through DOT1 L/H3 K79 me2-SIRT-XPA signal pathway.