沉默SIRT2基因对急性粒细胞白血病细胞增殖的影响

Effect of SIRT2 knock-down on proliferation of acute myeloid leukemia cell lines

目的 SIRT2是组蛋白去乙酰化酶家族成员之一,其参与多种肿瘤的发生和发展。本研究旨在探讨SIRT2基因在人急性粒细胞白血病细胞NB4和耐药细胞HL60/A增殖中的作用,分析SIRT2对于细胞周期的影响,寻找治疗急性粒细胞白血病的新靶点。方法 蛋白质印迹法检测SIRT2在急性粒细胞白血病细胞HL60/A和NB4中的表达,针对SIRT2mRNA不同的靶序列设计并合成shRNA,构建真核表达干扰载体,制备慢病毒并感染HL60/A和NB4细胞,用嘌呤霉素筛选出稳定表达shRNA的细胞,应用蛋白质印迹法验证干扰效率;采用改良MTT法检测沉默SIRT2基因前后HL60/A和NB4细胞增殖的改变;用流式细胞术检测沉默SIRT2基因前后细胞周期的变化。结果 在HL60/A和NB4细胞中SIRT2蛋白表达水平均增高,F=43.22,P=0.002。用慢病毒感染HL60/A（F=184.9,P〈0.001）和NB4（F=354.7,P〈0.001）后,SIRT2基因的蛋白表达水平显著降低。MTT结果显示,与对照组细胞比较,48h沉默SIRT2基因的HL60/A（F=208.4,P〈0.001）和NB4（F=27.58,P〈0.001）细胞的生长速度变慢;于72h这种抑制作用更加明显,其中HL60/A的增殖速度明显变慢,F=555.9,P〈0.001。流式细胞术检测细胞周期结果显示,G0～G1期百分比,与对照组HL60/A/con（27.62±1.01）%相比,干扰组HL60/A/sh1（37.14±0.03）%（F=7.61,P〈0.001）和HL60/sh2（37.51±3.63）%（F=3.89,P=0.009）比例显著增加;与对照组NB4/con（27.49±0.50）%相比,干扰组NB4/sh1（38.26±1.93）%（F=8.02,P=0.007）和NB4/sh2（35.23±1.11）%（F=3.50,P〈0.001）比例显著增加;S期细胞百分比,与对照组HL60/A/con（51.94±1.15）%相比,干扰组HL60/A/sh1（40.24±0.66）%（F=3.34,P〈0.001）和HL60/sh2（42.73±2.76）%（F=10.33,P=0.004）比例显著降低;与对照组NB4/con（50.65±0.23）%相比,干扰组NB4/sh1（39.58±1.11）%（F=3.02,P〈0.001）和NB4/sh2（43.82±1.33）%（F=10.42,P=0.011）比例显著降低;G2～M期细胞比例无明显变化。结论 干扰SIRT2基因的表达可使细胞周期阻滞在G0～G1期,从而抑制急性粒细胞白血病细胞HL60/A和NB4的增殖。

OBJECTIVE SIRT2 is a family member of histone deacetylases,involving in a wide range of cancers oc-currence and progression. We aimed to investigate the effect of SIRT2 gene on the proliferation and cell cycle distribution of human acute myeloid leukemia cell line NB4 and multidrug resistance cell line HL60/A, to provide the basis for new therapeutic target of AML. METHODS The expression of SIRT2 in HL60/A and NB4 cell lines was analyzed by Western blot. The shRNA targeting different specific sequences of the SIRT2 mRNA was designed and chemically synthesized. The shRNA vectors were constructed and were transfected respectively into 293T with packaging vectors to produce lentivirus. Thereafter, HL60/A and NB4 cells were infected with lentivirus and selected with puromycin to acquire stable cell lines expressing shRNA. The inhibitory efficiency of SIRT2 gene was measured by western blot. The effect of knock-down SIRT2 on the proliferation of HL60/A and NB4 cells was detected with MTT method, and cell cycle distribution was tested by flow eytometry. RESULTS SIRT2 was overexpressed in HL60/A and NB4 cell lines（F=43.22, P=0.002）. The protein expression level of SIRT2 in HL60/A（F=184.9,P〈0.001） and NB4（F=354.7,P〈0.001）cells was significantly reduced after infected with lentivirus. MTT assay showed that, compared with the control groups, the proliferation capacity of SIRT2 knock-down cells HL60/A （F = 208.4, P〈0. 001 ） and NB4 （F = 27.58, P〈0.001 ） was significantly inhibited at 48 h. The inhibition effect was more obvious, and the growth rate of HL60/A was significantly slower in 72 h（F=555.9, P〈0. 001）. The cell cycle tested by flow cytometry displayed that the proportion of Go -G1 phase cells was significantly increased in HL60/A/shl（37.14±0.03）%（F=7.61 ,P〈0. 001）, HL60/sh2（37.51± 3.63）%（F=3. 89,P=0. 009） compared with HL60/A/con（27. 62±1. 01）% and in NPA/shl（38. 26 ± 1. 93）% （F=8.02,P=0. 007）, NB4/sh2（35.23＋1.11）%（F=3.50,P〈0. 001）compared with NB4/con（27.49±0.50）%. The proportion of S phase cells was obviously reduced in HL60/A/shl （40.24! 0.66）% （F= 3.34, P〈0. 001）, HL60/sh2 （42.73±2.76） % （F=10.33,P=0. 004）compared with HL60/A/con（51.94±1.15）% and in NB4/shl（39.58＋1.11）% （F=3.02,P〈0. 001）, NB4/sh2（43. 82±1. 33）% （F= 10.42,P=0. 011） compared with NB4/con（50.65±0.23）%. However, there was no significant difference in the proportion of cells of G2-M phase. CONCLUSION The shRNA targeting SIRT2 can effectively inhibit the proliferation of human acute myeloid leukemia cell lines HL60/A and NB4 by making cell cycle arrest in G0-G1 phase.