沉默 DNMT1基因对 Molt-4细胞增殖、凋亡及组蛋白调控的影响

摘要

Aim To investigate the effect of small in-terfering 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 LipofectamineTM 2000. The DNMT1 mRNA and protein level were detected by RT-PCR and Western blot. Cell proliferation was de-termined 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 concentra-tion-dependent manner. DNMT1 siRNA suppressed cells proliferation and induced apoptosis in Molt-4 cells. 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 - 1 for 24 hours, P < 0. 05. The expres-sion of Bcl-2, procaspase-3 was suppressed and P15 was promoted after transfecting of DNMT1 siRNA. DN-MT1 siRNA downregulated histone methylated H3K9 and upregulated histone methylated H3K4. The altera-tion of histone acetylation of H3 was not seen. Conclu-sion 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 in-duces cell apoptosis in Molt-4 cell line.%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 -1 DN-MT1 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 siR-NA 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。