Effective siRNA targets screening for human telomerase reverse transcriptase.
OBJECTIVE
To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Xia
Yun
Y
Beijing Institute of Radiation Medicine, No. 27 Taiping Road, Beijing 100850, China.
Lin
Ru-Xian
RX
Zheng
Su-Jun
SJ
Yang
Ying
Y
Bo
Xiao-Chen
XC
Zhu
Dao-Yin
DY
Wang
Sheng-Qi
SQ
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D019098:Telomerase