沉默DNMT1基因对骨髓瘤细胞SOCS-1基因甲基化的影响  

Effects of DNMT1 Gene Silencing on Methylation of SOCS-1 Gene in Myeloma Cells

作　　者：王昭[1] 牛小青[1] 周雯雯[1] 鹿全意[1] 

机构地区：[1]厦门大学附属中山医院血液科福建医科大学教学医院,福建厦门361004

出　　处：《中国实验血液学杂志》2015年第3期713-717,共5页Journal of Experimental Hematology

摘　　要：目的:探讨DNA甲基化转移酶1(DNA methyltransferase,DNMT1)基因沉默对多发性骨髓瘤RPMI8226细胞内细胞因子信号转导抑制分子1(suppressor of cytokine signaling,SOCS-1)甲基化的影响。方法:应用靶向沉默DNMT1基因的短发卡RNA(RNA-Short hairpin RNA,shRNA)通过脂质体(lipofectamineTM2000)介导转染骨髓瘤细胞RPMI8226,应用RT-PCR和Western blot方法分别检测转染前后DNMT1基因和蛋白表达,应用甲基化特异性PCR检测转染前后骨髓瘤细胞SOCS-1基因甲基化水平的变化。结果:转染后骨髓瘤细胞DNMT1 mRNA表达水平(0.176±0.004)和蛋白表达水平(0.065±0.014)均明显低于阴性对照组(0.956±0.033,0.415±0.027),其差异具有统计学意义(P<0.01)。转染后SOCS-1基因甲基化水平较转染前明显降低。结论:利用shRNA能靶向沉默DNMT1基因的表达,并可以有效逆转SOCS-1基因高甲基化状态。Objective： To investigate the effect of DNA methyhransferase I（DNMT1 ） gene silencing on methylation of suppressor of cytokine signaling （SOCS-1） in multiple myeloma RPMI 8226 cells. Methods： Recombinant plasmid pshRNA-DNMT1 was transfected into multiple myeloma RPMI 8226 cells by lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of DNMT1 in RPMI 8226 cells respectively before and after transfection. Methylation-specific polyrnerase chain reaction was used to detect the methylation level change of SOCS-1 gene in RPMI8226 cells transfected. Results： DNMT1 targeted short hairpin RNA （shRNA） was successfully inserted into the plasmid vector pshRNA. RT-PCR results showed that the relative rnRNA expression level of DNMTI gene in RPMI 8226 cells transfected with pshRNA was 0. 176 ± 0. 004 which was significantly lower than that in cells transfected by empty vector（0.956 ± 0.033, P 〈 0.01 ）. Western blot analysis showed that the relative expression level of DNMT1 protein of RPMI 8226 cells transfected by pshRNA was 0.065 ± 0. 014, which was significantly lower than that in transfected cells by empty vector（0.415 ± 0.027 ） （P 〈 0.05 ）. These results indicated that the recombinant plasmid pshRNA could effectively knock down the expression of DNMT1 gene in RPMI 8226 cells. Methylation analysis showed that the methylation level of SOCS-1 gene was obviously reduced after transfection. Conclusion： DNMT1 gene in RPMI 8226 cell can be silenced by shRNA. DNMT1 gene silencing can significantly induce SOCS-1 gene hypomethylation, which indicates that DNMT1 may play an important role in the process of SOCS-1 hypermethylation.

关 键 词：多发性骨髓瘤 RNA干扰 甲基化转移酶1 SOCS-1基因 

分 类 号：R733.3[医药卫生—肿瘤]