摘要
目的探讨siRNA沉默人类急性髓细胞白血病HL-60细胞HDAC1基因表达后对细胞增殖、凋亡以及组蛋白甲基化、乙酰化修饰的影响。方法 LipofectamineTM2000为载体将HDAC1特异性siRNA转染入HL-60细胞后,RT-PCR检测HL-60细胞HDAC1mRNA表达,MTS法绘制细胞生长曲线,流式细胞术分析细胞凋亡,Western Blot检测HDAC1的表达及其对凋亡相关蛋白及组蛋白甲基化、乙酰化表达的影响。结果 HDAC1siRNA转染HL-60细胞后可沉默该基因表达;抑制HL-60细胞增殖,呈量效和时效关系;诱导HL-60细胞凋亡,经0,30,60,120nmol/L HDAC1siRNA处理24h后,细胞凋亡率分别为(1.43±0.19)%,(14.4±3.1)%,(57.7±4.2)%,(68.7±1.6)%;与空白对照组(1.43±0.19)%比较,差别具有统计学意义(P<0.05);且HDAC1基因沉默后,可下调Bcl-2,Procaspase-9,Procaspase-3,c-Myc的表达;上调组蛋白H3,H3K9,H3K14,H3K27乙酰化水平,下调H3K9甲基化水平,对H4乙酰化无明显影响。结论沉默HDAC1基因表达后可能通过上调与基因转录激活有关的组蛋白H3,H3K14,H3K9,H3K27乙酰化水平,下调与基因转录抑制有关H3K9甲基化水平,从而抑制细胞增殖,诱导细胞凋亡。
Objective To investigate the effect of silence histone deacetylasesl （HDAC1） gene by the technology of RNA interference on the proliferation, apoptosis and histone modulation in HL-60 cell line. Methods The optimal segment targeting HDAC1 gene was designed and transfected into HL-60 cells by LipofectamineTM2000. The HDAC1 mRNA and protein were detected by RT-PCR and Western blot. Growth inhibition was determined by MTS. Cell apoptosis was measured by flow cytometry. The expression of Bcl-2, procaspase-9, procaspase-3 and c-Myc,and the expression of histone methylation of H3K9 and histone acetylation of H3, H4,H3Kg, H3K14 and H3K27 were detected by Western blot.Results HDAC1 mRNA was markedly suppressed by the siRNA targeting HDAC1 in a concentration-dependent manner. HDAC1 siRNA suppressed the proliferation of HL-60 cells in a concentration- and time-dependent manners. HDAC1 siRNA induced cells apoptosis. Apoptotic rate was （1.43 ±0.19）%,（14.4±3, 1）%,（57.7±4.2）%,（68.7±1.6）%after transfected with HDAC1 siRNA at 30, 60, 120 nmol/L for 24 hours. It down-regulated the expression of Bcl-2, proCaspase9, proCaspase3 and c-Myc. HDAC1 siRNA upregulated histone acetylation o{ H3, H3Kg, H3K14 and H3K27, and down regulated histone methylation of H3K9. The change of histone acetylation of H4 was not seen. Conclusion HDAC1 siRNA inhibits the proliferation and induces apoptosis with concentration-dependent and time-dependent in HL-60 cell line. It upregulates histone acetylation of H3, H3K9, H3K14 and H3K27, and down-regulates histone methylation of H3K9. It doesn＇t affect histone acetylation of H4. HDAC1 might be a target of gene therapies for leukemia.
出处
《福建医科大学学报》
2013年第1期11-16,共6页
Journal of Fujian Medical University
基金
卫生部科学研究基金-福建卫生教育联合攻关计划项目(wkj2008-2-55)
福建省自然科学基金(2012J01420)
福建省卫生厅医学创新课题(2012-CX-32)
漳州市科学研究发展计划基金资助项目(20100080)
