Inhibitory effects of siRNA expression vector on the growth and hTERT gene expression of MCF-7 cells / 解放军医学杂志

ABSTRACT

Objective To explore the inhibitory effects of the vector on the growth of mammary adenocarcinoma cell MCF-7 and human telomerase reverse transcriptase(hTERT) gene expression in MCF-7 cells,by the way of the construction of the RNA interference(RNAi) expression vector of hTERT gene.Methods An interference target sequence TGT TCA GCG TGC TCA ACT A,which aimed at hTERT gene,was designed and the small interfering RNA(siRNA) expression plasmid pGenesil-hTERT was constructed by means of recombination.The recombinant plasmid was transfected into the MCF-7 cells by LipofectamineTM 2000 after the identification of the recombinant expression vector pGenesil-hTERT in the light of electrophoresis after digestion and DNA sequencing,then RT-PCR analysis was used to detect the gene expression,and Western blotting analysis was used herewith to detect the protein expression of hTERT,and the growth of MCF-7 cells was tested by MTT assay.Results The results of analysis and the sequencing identification confirmed that the target sequence was inserted into the predicted site precisely,and the recombining plasmid was successfully constructed.RT-PCR and Western blotting analysis showed that hTERT gene expression and protein expression of pGenesil-hTERT group were obviously decreased in MCF-7 cells,and the growth of MCF-7 cells of pGenesil-hTERT group was also inhibited.Conclusion The endogenous hairpin siRNA produced by DNA plasmid is capable of mediating hTERT gene inhibition in MCF-7 cells effectively,and furthermore inhibiting the cells growth and decreasing the speed of cells proliferation.This research may act as the experiment evidence to the gene therapy of cancer.