Objective: To investigate the effect of silencing LSD1 gene by RNA interference on the proliferation, apoptosis on human lymphocytic leukemia Jurkat cell line and its mechanism.
Methods: The hairpin- like oligonucleotide sequences targeting LSD1 gene was transfected into Jurkat cells by lipofectamine(TM) 2000. The LSD1 mRNA and protein were detected by RQ- PCR and Western blot. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of Bcl-2, Bax, procaspase- 3, and histone H3K4me, H3K4me2, H3K4me3, Act- H3, H3K9me were detected by Western blot.
Results: LSD1 mRNA was markedly suppressed by the shRNA targeting LSD1. LSD1 shRNA suppressed the proliferation and induced cells apoptosis of Jurkat cells. The cell apoptotic rate was (41.34±3.58)%, (3.45±1.54)%, (1.76±0.52)% in LSD1 shRNA, Neg-shRNA and Blank respectively, the difference among them was statistically significant (P<0.05). LSD1 shRNA down- regulated the expressions of Bcl- 2 and procaspase- 3, and up- regulated the expression of Bax. The methylation of H3K4me1, me2 and acetylation of Act- H3 improved without change of the methylation of H3K4me3.
Conclusions: Deplete of LSD1 gene maybe through modifying the methylation of histone H3K4 to promote the cell apoptosis and inhibit cell growth in Jurkat cell line.
目的: 探讨RNA靶向沉默LSD1基因后对急性T淋巴细胞白血病细胞株Jurkat细胞增殖、凋亡的影响及其可能的作用机制。
方法: 将LSD1短发夹RNA（shRNA）真核表达载体经脂质体转染入Jurkat细胞，采用RQ-PCR及Western blot方法观察LSD1 mRNA及蛋白表达；采用MTT法观察细胞增殖情况，采用流式细胞仪检测细胞凋亡情况，Western blot方法检测凋亡相关蛋白Bcl-2、Bax、procaspase-3的表达及组蛋白H3K4me、H3K4me2、H3K4me3、Act-H3水平。
结果: LSD1 shRNA转染Jurkat细胞后，LSD1 mRNA、蛋白表达均下调（P<0.05）；LSD1 shRNA组48 h细胞增殖率低于转染阴性质粒（Neg-shRNA）组和对照组（P<0.05），LSD1 shRNA组细胞凋亡率[（41.34±3.58）%]高于NegshRNA组[（3.45±1.54）%]和对照组[（1.76±0.52）%]（P<0.05）；凋亡抑制蛋白Bcl-2、凋亡效应蛋白procaspase-3的表达下调，Bax表达上调，H3K4me、H3K4me2、Act-H3表达上调，而H3K4me3水平未见明显变化。
结论: 沉默LSD1基因可抑制Jurkat细胞增殖，激活凋亡相关蛋白诱导细胞凋亡，机制可能与调节组蛋白H3K4甲基化水平有关。