急性单核白血病细胞系THP-1细胞MLL-AF9融合基因沉默对p27表达的影响

Effect of MLL-AF9 fusion gene silence of acute monocytic leukemia cell line THP-I on cyclin-depend- ent kinase inhibitor p27 expression

目的研究急性单核细胞白血病细胞系THP-1细胞MLL-AF9融合基因沉默对p27表达及转录调控的影响。方法选择THP-1细胞特有的MLL-AF9融合基因为靶基因，设计并合成MLL-AF9小干扰RNA（siRNA）片段。应用脂质体转染方法，将MLL-AF9 siRNA导入细胞，通过流式细胞术检测siRNA转染效率，用RT-PCR法比较转染前后MLL-AF9 mRNA表达水平的变化，Western blot检测MLL-AF9蛋白水平沉默效果，比较细胞周期抑制蛋白p27表达变化，利用染色质免疫沉淀（Chromatin Immunoprecipitation，CHIP）技术研究MLL-AF9融合蛋白是否与p27启动子结合。结果转染MLL-AF9 siRNA后THP-1细胞MLL-AF9 mRNA相对表达水平（0．31±0．07）较对照组（1．25±0．13）明显受抑（P〈0．叭），p27mRNA表达水平（0．84±0．12）较对照组（0．35±0．03）明显上调（P〈0．01），应用ChIP结合PCR扩增的方法分析发现MLL-AF9融合蛋白可与THP-1细胞p27基因启动子区域的DNA片段结合。结论MLL-AF9融合基因沉默后可使p27表达上调，其机制可能为MLL-AF9基因沉默解除了MLL-AF9融合蛋白与p27启动子的结合，从而恢复p27基因的表达。

Objective To explore the effect of MLL-AF9 fusion gene silence on p27 expression and transcription regulation in THP-1 cells. Methods Small interference RNA （siRNA） fragments targeting THP-1 cells specific MLL-AF9 fusion gene were designed and constructed, and transfected into THP-1 by li- pofectamine, Flow cytometry was used to detect siRNA transfection efficiency. The level of MLL-AF9 mRNA expression was examined by RT-PCR and the expression of MLL-AF9 and p27 protein was detected by Western blot. Chromatin immunoprecipitation （CHIP） assay was used to confirm whether MLL-AF9 binds to the p27 promoter in THP-1 cell. Results SiRNA transfection efficiency was （69.1±1.8） %. The level of p27 expression was up-regulated at both mRNA[（0.84±0.12） vs （0.35±0.03） of control group] and protein levels after MLL-AF9 expression was significantly inhibited in siRNA-transfected cells （0.31±0.07） compared with that in the controls （1.25±0.13 ） （P 〈 0.01 ）. M LL-AF9 fusion protein binded to DNA fragment of p27 gene promoter region in THP-1 cell. Conclusion MLL-AF9 fusion gene silence up-regulates p27 gene expression, and the mechanism maybe the recovery of p27 gene expression due to MLL-AF9 fusion protein binding to p27 promoter.