Treatment Of Human Breast Cancer Cells With Antisense RNA To IGF-IR Inhibits The Expression Of Endogenous IGF-IR In Vitro

Objective: To study the inhibition of exogenous antisense RNA of type I insulin-like growth factor receptor (IGF-IR) to endogenous expression of IGF-IR in human breast cancer cells.Methods: Design a pair of primers which contain two restriction site, BamH1 and Hindâ…¢ and hIGF-IR cDNA fragments were amplified by RT-PCR. The purified PCR products and empty plasmid pcDNA3.1(-) were digested by BamHl and Hindlll, ligated , transformed ,indentified and positive recombinants were acquired, then MCF-7 human breast cancer cells were transfected with recombined vector, G418 was used to selected for cells that were stably transfected with recombined vector. Transfected and nontransfected cells were screened for expression of endogenous IGF-IR mRNA and protein.Results: The two fragments from recombined hIGF-IR antisense eukaryotic expression vector by BamHl and Hindlll represented 700bp and 5.4kb by agarose electrophoresis, PCR showed positive fragment which was about 700bp long, sequence analysis showed the same sequence as expected. Recombined insulin-like growth factor I receptor antisense eukaryotic expression vector (pIGF-IR/As) has been constructed successfully. The expression of IGF-IR mRNA and protein were decreased after antisense IGF-IRcDNA transfected, detected by RT-PCR, Western blot.Conclusion: The recombined pIGF-IR/As can stably express antisense RNA which can inhibits the expression of endogenous IGF-IR obviously.