摘要
目的构建表达靶向抑制垂体瘤转化基因1(PTTG1)基因表达的RNA干扰载体microRNA,导入人脑胶质瘤细胞株U251中,研究靶向抑制PTTG1基因对U251细胞的凋亡诱导作用。方法以PTTG1为靶基因,根据pcDNATM6.2-GW/EmGFP-miR载体要求,设计针对PTTG1的microRNA序列,脂质体转染法将重组质粒转入U251细胞,采用荧光定量多聚酶链反应(PCR)以及Western blot分别从mRNA和蛋白水平检测干扰效果;膜联蛋白V与碘化丙啶(annexinV/PI)双色标记的流式细胞仪法检测microRNA诱导的细胞凋亡,碘化丙锭(PI)染色检测细胞周期阻滞。结果实时荧光定量PCR以及Western blot检测显示,PTTG1基因的mRNA转录水平以及蛋白水平的表达均得到显著抑制;转染后,流式细胞仪检测分析显示,PTTG1基因表达被抑制后,U251细胞凋亡率明显增高,细胞周期出现明显的G2/M阻滞。结论靶向PTTG1基因的重组microRNA干扰载体pcDNA6.2-GW/EmGFP-miR-PTTG1介导的RNAi显著靶向抑制了PTTG1基因在人脑胶质瘤细胞中的表达,并明显地诱导了脑胶质瘤细胞发生凋亡和G2/M期细胞周期阻滞。
Objective To explore the effect of RNA interference mediated pituitary tumor transforming gene 1（PTTG1） silencing on proliferation and apoptosis of human glioma U251 cells.Methods MicroRNAs（miRNA） targeting PTTG1 gene was designed and DNA template was synthesized,then miRNA was obtained by in vitro transcription.After miRNA was transfected into U251 cells with Lipofectamine,the proliferation of U251 cells was detected by MTT colorimetry,and AnnexinV/PI double staining by flow cytometry analysis was used to evaluate apoptosis.The change of cell cycle was analyzed by flow cytometry（FCM）.The expression of PTTG1 on mRNA and protein level was analyzed by real-time quantitative RT-PCR and Western blotting,respectively.Results The expression of PTTG1 at both RNA level and protein level was significantly down-regulated as validated by real-time quantitative PCR and Western blot.Flow cytometry analysis revealed glioma cell line U251 suffered a notable apoptosis and G2/M arrest after RNA interference mediated by miRNA targeting PTTG1.Conclusion RNA interference（RNAi） mediated by the miRNA expression vector pcDNATM6.2-GW/EmGFP-miR-PTTG1 can significantly down-regulate the expression of PTTG1,induce remarkable apoptosis and G2/M cell cycle arrest in glioma cell line U251 in vitro.
出处
《中华神经外科疾病研究杂志》
CAS
2012年第1期28-31,共4页
Chinese Journal of Neurosurgical Disease Research
