[1]赵先兰)△,孟志英),饶燕玲),等.靶向DNA甲基转移酶1的siRNA表达载体的构建与筛选[J].郑州大学学报(医学版),2010,(04):600.
 [J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2010,(04):600.
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靶向DNA甲基转移酶1的siRNA表达载体的构建与筛选()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2010年04期
页码:
600
栏目:
论著
出版日期:
2010-07-30

文章信息/Info

作者:
赵先兰1)△孟志英1)饶燕玲1)孙亚兰1)姜国忠2)刘红涛3)
1)郑州大学第一附属医院妇产科 郑州 450052
2)郑州大学基础医学院病理学教研室 郑州 450001
3)郑州大学生物工程系 郑州 450001
关键词:
DNA甲基转移酶1RNA干扰HeLa细胞构建载体筛选
文献标志码:
A
摘要:
目的:构建针对DNA甲基转移酶(dnmt1)基因的小干扰RNA(small interference RNA, siRNA)表达载体,以用于后续的 RNA干扰研究。方法:针对人 dnmt1的mRNA序列,设计并合成6条编码siRNA的寡核苷酸片段, 经退火成互补双链,克隆至载体pSilencer3.1H1中构建3个重组体,分别命名为pshRNAdnmt1A、pshRNAdnmt1B和pshRNAdnmt1C, 并进行测序鉴定。再利用无内毒素的大提质粒试剂盒制备无内毒素的pshRNAdnmt1A、B和C,分别通过脂质体介导转染宫颈癌HeLa细胞,以未转染和转染空载体者为对照。采用RTPCR 和Western blot检测转染细胞dnmt1 mRNA及蛋白表达情况。结果:成功构建了表达靶向dnmt1的siRNA重组载体;转染pshRNAdnmt1C对HeLa细胞dnmt1基因的表达几乎无影响。pshRNAdnmt1A、B均能有效沉默HeLa细胞中dnmt1基因的表达,其dnmt1 mRNA表达的抑制率分别为21.8%和69.7%,蛋白表达的抑制率分别为27.9%和73.2%,以pshRNAdnmt1B的干扰效果最佳。结论:成功构建并筛选出了有效的dnmt1 siRNA表达载体。

参考文献/References:

[1]Novina CD, Sharp PA. The RNAi revolution[J]. Nature, 2004,430(6 996):161
[2]Kim D, Rossi J. RNAi mechanisms and applications[J]. Biotechniques, 2008, 44(5):613
[3]Dalmay T. MicroRNAs and cancer[J]. J Intern Med, 2008, 263(4):366
[4]Brummelkamp TR, Bernards R, Agami R.A system for stable expression of short interfering RNAs in mammalian cells[J]. Science, 2002, 296(5 567):550
[5]Abreu PA, DellamoraOrtiz G, LeáoFerreira LR, et al. DNA methylation: a promising target for the twentyfirst century[J]. Expert Opin Ther Targets, 2008, 12(8):1 035
[6]Svedruzic ZM. Mammalian cytosine DNA methyltransferase Dnmt1: enzymatic mechanism, novel mechanismbased inhibitors, and RNAdirected DNA methylation[J].Curr Med Chem,2008,15(1):92
[7]Luczak MW, Jagodziński PP. The role of DNA methylation in cancer development[J].Folia Histochem Cytobiol,2006,44(3):143
[8]Nagai M, Nakamurama A, Makino R, et al. Expression of DNA(5eytosin)methyltransferase (DNMTs) in hepatocellular carcinomas[J].Hepatol Res,2003,26(3):186
[9]Etoh T, Kanai Y, Ushijima S, et al. Increased DNA methyltranseferase Ⅰ(DNMTⅠ) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpG islands in gastric cancers[J].Am J Pathol,2004,164(2):689
[10]Ehrlich M, Woods CB, Yu MC, et al. Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors[J].Oncogene,2006,25(18):2 636
[11]Xiong Y, Dowdy SC, Xue A, et al. Opposite alterations of DNA methyltransferases gene expression in endometrioid and serouse endometrial cancers[J]. Gynecol Oncol,2005,96(3):601

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