靶向抑制 DNMT1对人脑胶质瘤细胞增殖的影响

摘要

Objective To transfect the human glioblastoma cells in vitro with the siRNA sequence targeting to DNA methyltransferase 1(DNMT1)gene,and investigate its effect on proliferation of human glioblastoma cells. Methods The experimental group was transfected by siRNA-DNMT1 sequence and the control group was given the siRNA negative sequence. The expression of DNMT1 gene was confirmed by qRT-PCR. The expression of DNMT1,PCNA and Cyclin D1,which were usually used as cell proliferation markers,was analyzed by Western blot. Cell survival and proliferation rate were determined by MTT and colony formation assay. Results Compared with the control group,qRT-PCR results showed that DNMT1 mRNA level was significantly decreased in DNMT1 siRNA transfected cells(P < 0. 01). The protein expression levels of DNMT1,PCNA and Cyclin D1 were dramatically reduced in the experimental group as detected by Western blot( P < 0. 01). MTT and colony formation assay results suggested that,the experimental group had a much lower cell survival(P < 0. 05)and proliferation rate(P < 0. 01). Conclu-sion The expression of DNMT1 gene of human glioblastoma cells in vitro is decreased by siRNA sequence targeting to DNMT1 gene,and the proliferation of human glioblastoma cells is also inhibited.%目的：应用靶向 DNA 甲基转移酶1（DNMT1）基因的小干扰 RNA（siRNA）重组质粒来转染体外培养的人脑胶质瘤细胞，观察其对胶质瘤细胞增殖活性的影响。方法实验组予以 siRNA-DNMT1序列进行转染，对照组仅予以 siRNA-阴性对照序列。应用 qRT-PCR 分析 DNMT1基因表达变化， Western blot 法分析 DNMT1、PCNA 和 Cyclin D1蛋白表达变化，MTT 法检测细胞增殖生长能力，细胞克隆法分析细胞增殖克隆形成能力。结果与对照组比较，qRT-PCR 结果表明实验组 DNMT1 mRNA 表达明显减少（ P <0.01）；Western blot 法表明实验组 DNMT1、PCNA 和 Cyclin D1蛋白表达水平明显降低（P <0.01）；MTT 法检测表明实验组中活细胞数明显低于对照组（P <0.05）；细胞克隆法表明实验组细胞的增殖克隆形成能力明显低于对照组（ P <0.01）。结论靶向 DNMT1基因的 siRNA 重组质粒可减少人脑胶质瘤细胞内 DNMT1基因的表达，从而抑制胶质瘤细胞的增殖。