摘要
背景与目的:DNA甲基转移酶1(DNAmethyltransferase1,dnmt1)基因在多种肿瘤细胞中表达量相当高,而在正常成人细胞中则低表达,因此dnmt1基因的高表达与肿瘤的发生有密切的关系。本研究观察以dnmt1基因为靶基因的重组质粒对胃癌细胞株AGS细胞周期、增殖及凋亡的影响。方法:设计短发夹状RNA(shorthairpinRNA,shRNA)的寡核苷酸片段,再克隆至载体pTZU6+1中构建重组体pshRNA-dnmt1,转染胃癌细胞株AGS。实验分为空白对照组(仅予脂质体)、pTZU6+1组(予空质粒pTZU6+1)和pshRNA-dnmt1组(予以重组质粒pshRNA-dnmt1)3组。Westernblot检测dnmt1蛋白水平变化;RT-PCR法评估mRNA水平;流式细胞技术分析细胞周期的变化;MTT法检测细胞生长情况;吖啶橙/溴乙锭双染色(AO/EB)法、电镜和TUNEL观察细胞凋亡情况。结果:成功构建重组质粒pshRNA-dnmt1,并经序列分析得到确认。pshRNA-dnmt1转染AGS细胞后24h出现dnmt1蛋白表达量减少,24、48、72h抑制率分别为28.24%、68.54%、81.47%。转染pshRNA-dnmt1后24、48、72h对AGS细胞中dnmt1mRNA的抑制率分别为21.63%、52.97%、72.06%。转染后72hS期细胞由空白对照组的(36.58±1.76)%降至pshRNA-dnmt1组的(18.54±6.59)%;G2/M期细胞由空白对照组的(6.18±0.32)%升至pshRNA-dnmt1组的(18.53±1.42)%,均有显著性差异(P<0.05)。转染后24、48、72h细胞存活率分别为79.49%、51.63%和39.16%。AO/EB法、电镜和TUNEL均可见大量典型的凋亡和坏死细胞。结论:重组质粒pshRNA-dnmt1能特异有效地抑制AGS细胞内dnmt1的表达、抑制细胞增殖、促进细胞凋亡,为肿瘤的基因治疗提供依据。
BACKGROUND ＆ OBJECTIVE. Dnmt1, a major DNA methyltransferase gene, is highly expressed in many cancers and lowly expressed in normal adult cells, therefore, its overexpression is closely related to tumorigenesis. This study was to assess effects of dnmt1 gene silencing on cell cycle, proliferation, and apoptosis of gastric cancer cell line AGS. METHODS. The eukaryotic expression plasmid pshRNA-dnmt1, containing the sequence of short hairpin RNA （shRNA） targeting dnmt1, was constructed and transfected into AGS cells. PBS-treated cells and pTZU6＋1- transfected cells were set as control. The expression levels of dnmt1 were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. Cell cycle was analyzed by flow cytometry; cell survival was analyzed by MTT assay; cell apoptosis was evaluated by AO/EB double staining, electron microscopy, and TUNED RESULTS. pshRNA-dnmt1 targeting dnmt1 was successfully constructed, and confirmed by sequencing. Relative to control, 24, 48, and 72 h after transfection of pshRNA-dnmt1, the inhibitory rates of dnmt1 protein levels in AGS cells were 28.24%, 68.54%, and 81.47%, respectively, and those of dnmt1 mRNA levels were 21.63%, 52.97%, and 72.06%, respectively. The growth of AGS cells was suppressed 72 h after transfection. S phase cells were reduced from （36.58±1.76）% to （18.54±6.59）% （P〈0.05）, and GJM phase cells were increased from （6.18±0.32）% to （18.53±1.42）% （P〈0.05）. Cell survival rates were 79.49%, 51.63%, and 39.16%, respectively, 24, 48, and 72 h after transfection of pshRNA-dnmt1. A lot of apoptotic and necrosis cells were observed after transfection. CONCLUSIONS. The recombinant plasmid pshRNA-dnmt1 can efficiently and specifically inhibit the expression of dnmt1 gene and the proliferation of AGS cells, and induce cell apoptosis. It provides evidence for gene therapy of human cancers.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2006年第3期308-314,共7页
Chinese Journal of Cancer
基金
重庆市自然科学基金(No.CSCT2005BB5311)
国家自然科学基金~~
作者简介
通讯作者：陈道荣 Tel：86-23-68122549 E-mail：cdrcdrcn@yahoo．com．cn
