CAJ | 학술논문

目的观察LSD1基因对人类急性T淋巴母细胞性白血病Molt-4细胞增殖和凋亡的影响。方法设计并筛选出针对LSD1基因的最佳siRNA片段,将其转染入Molt-4细胞后,MTS法观察LSD1 siRNA对Molt-4细胞增殖的影响；流式细胞术分析细胞凋亡；Western blot检测LSD1 siRNA作用后组蛋白H3 K4、H3 K9甲基化及组蛋白H3乙酰化状态,以及p15、DNA甲基化酶1( DNMT1)和凋亡相关蛋白 Bcl-2、pro-caspase-3的表达变化。结果沉默 LSD1基因可抑制细胞增殖,LSD1 siRNA浓度为0、30、60、120 nmol·L-1作用48 h后,Molt-4细胞的增殖率分别为(99．65±1．21)%、(83．02±1．69)%、(65．72±2．16)%、(41．15±2．23)%,差异具有统计学意义( P<0．05)； LSD1 siRNA以60 nmol · L-1的浓度转染细胞0、24、48、72 h,增殖率分别为(99．86±1．35)%、(65．72±2．16)%、(48．26±1．92)%、(37．86±1．66)%,P<0．05,提示LSD1 siRNA可以抑制Molt-4细胞的增殖。 LSD1 siRNA 0、30、60、120 nmol·L-1作用48 h后,细胞凋亡率分别为(3．35±1．26)%、(12．16±1．74)%、(32．74±2．47)%、(54．64±2．58)%,P<0．05,凋亡率随着LSD1 siRNA浓度的增加逐渐上升；同时出现凋亡相关蛋白Bcl-2、procaspase-3的表达下降；LSD1 siRNA抑制LSD1蛋白及LSD1 mRNA,上调组蛋白H3K4一甲基化、二甲基化及组蛋白H3的乙酰化水平, H3 K4三甲基化、H3 K9甲基化水平无明显变化；LSD1 siRNA下调DNA去甲基化酶DNMT1的表达,上调 p15的表达。结论 LSD1 siRNA 能抑制 Molt-4细胞的增殖并诱导其凋亡,其机制可能与表观遗传学调控有关,有望成为白血病治疗的一个新的靶点。
목적관찰LSD1기인대인류급성T림파모세포성백혈병Molt-4세포증식화조망적영향。방법설계병사선출침대LSD1기인적최가siRNA편단,장기전염입Molt-4세포후,MTS법관찰LSD1 siRNA대Molt-4세포증식적영향；류식세포술분석세포조망；Western blot검측LSD1 siRNA작용후조단백H3 K4、H3 K9갑기화급조단백H3을선화상태,이급p15、DNA갑기화매1( DNMT1)화조망상관단백 Bcl-2、pro-caspase-3적표체변화。결과침묵 LSD1기인가억제세포증식,LSD1 siRNA농도위0、30、60、120 nmol·L-1작용48 h후,Molt-4세포적증식솔분별위(99．65±1．21)%、(83．02±1．69)%、(65．72±2．16)%、(41．15±2．23)%,차이구유통계학의의( P<0．05)； LSD1 siRNA이60 nmol · L-1적농도전염세포0、24、48、72 h,증식솔분별위(99．86±1．35)%、(65．72±2．16)%、(48．26±1．92)%、(37．86±1．66)%,P<0．05,제시LSD1 siRNA가이억제Molt-4세포적증식。 LSD1 siRNA 0、30、60、120 nmol·L-1작용48 h후,세포조망솔분별위(3．35±1．26)%、(12．16±1．74)%、(32．74±2．47)%、(54．64±2．58)%,P<0．05,조망솔수착LSD1 siRNA농도적증가축점상승；동시출현조망상관단백Bcl-2、procaspase-3적표체하강；LSD1 siRNA억제LSD1단백급LSD1 mRNA,상조조단백H3K4일갑기화、이갑기화급조단백H3적을선화수평, H3 K4삼갑기화、H3 K9갑기화수평무명현변화；LSD1 siRNA하조DNA거갑기화매DNMT1적표체,상조 p15적표체。결론 LSD1 siRNA 능억제 Molt-4세포적증식병유도기조망,기궤제가능여표관유전학조공유관,유망성위백혈병치료적일개신적파점。
Aim To observe the effect of the LSD1 gene on the proliferation and apoptosis of Molt-4 cells, a kind of human acute T-lymphoblastic leukemia cells. Methods siRNA fragment based on LSD1 gene was designed, filtered out and then transfected into Molt-4 cells. The effects of LSD 1 siRNA on Molt-4 cell prolif-eration were observed by the method of MTS. The cell apoptosis was analyzed by flow cytometry. The states of histone H3K4, H3K9 methylation, histone H3 acetyla-tion, p15, DNA methyltransferase 1 (DNMT1), and apoptosis-related proteins like Bcl-2 , procaspase-3 were evaluated by Western blot. Results Silencing LSD1 gene inhibited cell proliferation. Molt-4 cell pro-liferation rate was ( 99. 65 ± 1. 21 )%, ( 83. 02 ± 1. 69)%, (65. 72 ± 2. 16)%,and (41. 15 ± 2. 23)%respectively after the treatment of Molt-4 cells with 0 , 30, 60, 120 nmol·L-1 of LSD1 siRNA after 48 hours ( P < 0. 05 ) . Cell proliferation rate was ( 99. 86 ± 1. 35)%,(65. 72 ± 2. 16)%,(48. 26 ± 1. 92)%,and ( 37. 86 ± 1. 66 )% respectively after the transfection of Molt-4 cells with 60 nmol · L-1 of LSD1 siRNA after 0 , 24 , 48 , 72 hours ( P<0. 05 ) . Cell apoptosis rate was ( 3. 35 ± 1. 26 )%, ( 12. 16 ± 1. 74 )%, ( 32. 74 ± 2. 47 )%, ( 54. 64 ± 2. 58 )% respectively after transfection of LSD1 siRNA in indicated concentrations for 48 hours ( P <0. 05 ) . At the same time, the ex-pression levels of apoptosis-related proteins like Bcl-2 , procaspase-3 decreased. LSD1 siRNA inhibited LSD1 and LSD1 mRNA, and accumulated histone mono-, and di-methylation H3K4 and histone H3 acetylation. However, alteration of H3K4 trimethylation, H3K9 methylation was not detected. LSD1 siRNA downregu-lated DNA demethylase DNMT1 and upregulated p15 . Conclusions LSD1 siRNA can inhibit Molt-4 cell proliferation and induce apoptosis. Its mechanism may be associated with epigenetic regulation. In addition, it is expected to become a new target for leukemia treat-ment.