Construction of hTERT-siRNA expression vector and its inhibitory effects on the growth of MCF-7 cells and the activity of telomerase
- VernacularTitle:hTERT-siRNA表达载体的构建及对MCF-7细胞生长、端粒酶活性的抑制作用
- Author:
Zhihong ZHANG
;
Yongqiu ZENG
;
Qinglin SHUI
- Publication Type:Journal Article
- Keywords:
RNA interference;
siRNA expression vector;
genes,hTERT;
apoptosis
- From:
Medical Journal of Chinese People's Liberation Army
1981;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the RNAi expression vector of hTERT gene,and to investigate the inhibitory effects of the vector on the growth of mammary adenocarcinoma cell MCF-7 and the activity of telomerase.So as to present a new approach to the gene therapy for breast cancer which aim at inhibiting the telomerase activity.Methods An interference target sequence TGTTCAGCGTGCTCA ACTA which aimed at hTERT gene was designed and the siRNA expression plasmid pGenesil-hTERT was constructed by the means of recombination.Meanwhile a negative control recombinant-pGenesil-HK that did not aim at any gene was constructed by the same method.The recombinants expression vectors pGenesil-hTERT and pGenesil-HK were identified by electrophoresis after digestion and DNA sequencing.The two recombinants were transfected into the MCF-7 cells by lipofectamineTM 2000.The telomerase activity was tested by telomerase repeat sequence amplification and polyacrylamide gel electrophoresis.Flow cytometry was used to detect the cell apoptosis.Results The results of analysis and the sequencing identification confirmed that the target sequence was inserted into the predicted site precisely and the recombining plasmids were successfully constructed.The PAGE results showed that the mark traps of telomerase were obviously decreased and the telomerase activity of pGenesil-hTERT was obviously inhibited.The apoptotic rate of the pGenesil-hTERT group was significantly higher than that of the pGenesil-HK group(P
