Xia Yun, Lin Ru-Xian, Zheng Su-Jun, Yang Ying, Bo Xiao-Chen, Zhu Dao-Yin, Wang Sheng-Qi
Beijing Institute of Radiation Medicine, No. 27 Taiping Road, Beijing 100850, China.
World J Gastroenterol. 2005 Apr 28;11(16):2497-501. doi: 10.3748/wjg.v11.i16.2497.
To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence.
Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4 sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000. At 48-72 h after siRNAs transfection, MTT assay, RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively.
Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2 cells.
siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method, synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.
研究靶向不同hTERT序列的小干扰RNA（siRNA）的抑制作用，筛选有效的siRNA序列。
采用T7转录系统体外设计并合成5条靶向hTERT基因编码区和非编码区的双链siRNA。通过全长基因靶向技术筛选出siRNA4序列，其余siRNA序列随机选取。经乙醇沉淀纯化后，用Lipofectamine 2000将siRNA转染至人肝癌细胞（HepG2）。在转染siRNA后48 - 72小时，分别采用MTT法、RT-PCR和Western-blot检测siRNA对细胞生长、hTERT基因mRNA和蛋白表达水平的影响。
与对照细胞相比，用5条双链siRNA处理的细胞均呈现不同程度的细胞增殖抑制，且呈剂量依赖性。siRNA2和siRNA4对HepG2细胞中hTERT mRNA和蛋白表达具有明显的抑制作用。
靶向不同hTERT序列的siRNA对hTERT基因表达具有显著不同的抑制作用。通过全长基因靶向技术筛选的siRNA序列与随机筛选的其余siRNA序列具有相当的抑制效果，提示siRNA和反义寡核苷酸可能具有相同的有效靶位点。与化学合成方法相比，利用T7转录系统体外合成双链siRNA是一种快速、简便且廉价的方法，适用于筛选针对特定基因的高效siRNA靶向位点。
