SiRNA Inhibiting The Expression Of ER81(ETV1)and The Influence On The Growth Of The Human Breast Cancer Cell Line MDA-MB-231

Objective:Three siRNAs targeting against ER81 of human were designed by RNA interference(RNAi)technique and then inserted into pGenesil expression vecter to investigate if this expression system inhibit the expression of ER81 and promote the apoptosis of breast cancer cell line MDA-MB-231,and provide a new treatment approach for breast cancer.Methods:1.Three sequences targeting against ER81 and the scrambled siRNA, GAPDH-siRNA as contrast were inserted into pGenesill expression vector after screening,designing and synthesizing.The clone was identified by restriction enzyme digestion and DNA sequencing technique.2.Cell line MDA-MB-231 were divided into 7 groups:group recombinant plasmid ETV1A,group recombinant plasmid ETV1B,group recombinant plasmid ETV1C,group blank cells,group scrambled siRNA,group empty vector,group GAPDH-siRNA.Gene was stable transferred into human breast cancer MDA-MB-231 cell line using lipofectamine.Determination of efficiency of transcription and translation of ER81 was performed by semi quantitative RT-PCR,Western blotting.Cell apoptosis were measured by flow cytometry.Results:1.PGenesill expression vector was successfully constructed and the sequence of constructed recombinant plasmid was correct by restriction enzyme digestion and DNA sequencing.2.The expression of ER81 mRNA and protein were decreased markedly than control group by semi quantitative RT-PCR,Western blotting after PGenesil 1-ETV1A/ETV1B/ETV1C-siRNA transfected using lipofectamine,especially in group ETV1A and ETV1C.3.Flow cytometry detection of apoptosis rate in each group,the difference was not statistically significant.Conclusion:1.The pGenesill expression vector targeting anainst ER81 was successfully constructed.2.The PGenesil1-ETV1A/ETV1B/ETV1C-siRNA transfecting target cells MDA-MB-231 using lipofectamine has taken gene silencing effect remarkably,especially in group ETV1A and ETV1C.3.Flow cytometry detection of apoptosis rate in each group,the difference was not statistically significant.