沉默DNMT1基因对Molt-4细胞增殖、凋亡及组蛋白调控的影响  

Effect of Silencing DNMT1 gene on histone methylation modulation,cell proliferation and apoptosis in Molt-4 cell line

作　　者：陈淑瑜[1] 黄轶群[2] 游育红[3] 马旭东[2] 

机构地区：[1]漳州卫生职业学院药学系 [2]福建医科大学附属漳州市医院血液科,福建漳州363000 [3]福建医科大学药学院,福建福州350004

出　　处：《中国药理学通报》2016年第1期64-69,共6页Chinese Pharmacological Bulletin

基　　金：福建省引进重大项目计划基金(No 2012I2004);福建省自然科学基金资助项目(No 2012J01420);福建医科大学科学研究专项基金资助项目(重点类)(No 2013FZS13004Z);福建省医学创新课题(No 2012-CX-32);福建省中青年教师教育科研项目(No JB13364)

摘　　要：目的探讨siRNA沉默DNMT1基因对人急性T淋巴细胞性白血病Molt-4细胞增殖、凋亡及组蛋白调控的影响。方法将DNMT1特异性siRNA经LipofectamineTM2000转染Molt-4细胞后,应用RT-PCR检测Molt-4细胞DNMT1 mRNA表达,MTT法绘制细胞生长曲线;流式细胞术分析细胞凋亡。Western blot检测Bcl-2、procaspase-3、P15蛋白表达以及组蛋白甲基化、乙酰化状态的改变。结果 DNMT1 siRNA可沉默DNMT1基因的表达,并呈现浓度依赖性;抑制Molt-4细胞增殖,诱导细胞凋亡。经0、30、60、120 nmol·L-1DNMT1 siRNA作用24h后,细胞凋亡率分别为(4.27±1.42)%,(15.25±1.54)%,(35.63±2.54)%,(66.27±3.02)%,差异具有统计学意义(P<0.05)。DNMT1 siRNA可上调P15的表达;下调Bcl-2、procaspase-3的表达,下调组蛋白H3K9甲基化水平,上调组蛋白H3K4的甲基化水平,而组蛋白H3乙酰化水平无明显变化。结论 DNMT1 siRNA可以有效地抑制Molt-4细胞中DNMT1的表达,下调组蛋白H3K9甲基化水平,上调组蛋白H3K4的甲基化水平,从而抑制细胞增殖和诱导细胞凋亡。Aim To investigate the effect of small interfering RNA( siRNA) targeting DNMT1 gene on cell proliferation,apoptosis and histone modulation in acute lymphoid leukemia cell line,Molt-4. Methods The small interfering RNA targeting DNMT1 gene was transfected into Molt-4 cells by LipofectamineTM2000.The DNMT1 mRNA and protein level were detected by RT-PCR and Western blot. Cell proliferation was determined by MTT. Cell apoptosis was measured by Flow Cytometry. The expression of Bcl-2,procaspase-3,P15,histone methylation and histone acetylation was detected by Western blot. Results DNMT1 was suppressed by siRNA targeting DNMT1 in a concentration-dependent manner. DNMT1 siRNA suppressed cells proliferation and induced apoptosis in Molt-4cells. Apoptotic rate was( 4. 27 ± 1. 42) %,( 15. 25± 1. 54) %,( 35. 63 ± 2. 54) %,( 66. 27 ± 3. 02) %after transfecting with DNMT1 siRNA at 0,30,60,120 nmol· L- 1for 24 hours,P < 0. 05. The expression of Bcl-2,procaspase-3 was suppressed and P15 was promoted after transfecting of DNMT1 siRNA. DNMT1 siRNA downregulated histone methylated H3K9 and upregulated histone methylated H3K4. The alteration of histone acetylation of H3 was not seen. Conclusion DNMT1 siRNA suppresses DNMT1 efficiently in Molt-4 cells. The depletion of DNMT1 downregulates histone methylation of H3K9,and upregulates histone methylation of H3K4. It inhibits cell growth and induces cell apoptosis in Molt-4 cell line.

关 键 词：RNA干扰 DNMT1 组蛋白 乙酰化 甲基化 急性白血病 表观遗传学 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R329.25[医药卫生—基础医学]