Han Shiwei, Huang Yiqun, Zheng Ruiji
Department of Hematology, Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000, China.
Zhonghua Xue Ye Xue Za Zhi. 2016 Jan;37(1):56-60. doi: 10.3760/cma.j.issn.0253-2727.2016.01.011.
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.
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.
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.
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.
探讨RNA干扰沉默赖氨酸特异性去甲基化酶1（LSD1）基因对人淋巴细胞白血病Jurkat细胞株增殖、凋亡的影响及其机制。
采用脂质体2000将靶向LSD1基因的发夹状寡核苷酸序列转染至Jurkat细胞。采用实时定量聚合酶链反应（RQ-PCR）和蛋白质免疫印迹法检测LSD1 mRNA和蛋白表达。采用噻唑蓝（MTT）法检测细胞生长情况。采用流式细胞术分析细胞凋亡情况。采用蛋白质免疫印迹法检测B细胞淋巴瘤-2（Bcl-2）、Bax、半胱天冬酶原-3（procaspase-3）以及组蛋白H3K4单甲基化（H3K4me1）、H3K4二甲基化（H3K4me2）、H3K4三甲基化（H3K4me3）、乙酰化组蛋白H3（Act-H3）、H3K9甲基化（H3K9me）的表达。
靶向LSD1的短发夹RNA（shRNA）可显著抑制LSD1 mRNA表达。LSD1 shRNA可抑制Jurkat细胞增殖并诱导其凋亡。LSD1 shRNA组、阴性对照shRNA（Neg-shRNA）组和空白对照组细胞凋亡率分别为（41.34±3.58）%、（3.45±1.54）%、（1.76±0.52）%，差异有统计学意义（P<0.05）。LSD1 shRNA可下调Bcl-2和procaspase-3表达，上调Bax表达。H3K4me1、H3K4me2甲基化及Act-H3乙酰化水平升高，而H3K4me3甲基化水平无变化。
LSD1基因缺失可能通过改变组蛋白H3K4甲基化促进Jurkat细胞凋亡并抑制细胞生长。
