The Filtration Of The Breast Cancer Cell Inhibitory Peptide Aptamers In Nerve Growth Factor Signaling Pathway

Objective:To investigate the nerve growth factor signaling pathway in human breast cancer MCF-7 cells,to select the breast cancer cell proliferation inhibitory peptide aptamers and to find the potential therapeutic target for breadst cancer.Methods:Immunofluorescence and western blotting method to detect NGF and its receptor TrkA protein in MCF-7 cells;The use of cell counting kit-8(CCK-8) to detect the effect of different concentrations of NGF antagonist Ro 08-2750 and K252a on cell proliferation and survival;Western blotting to detect the changes of active-MAPK and p53 protein in MCF-7 cells being treated with different concentrations of Ro 08-2750;the use of flow cytometry to detect the apotosis as well as changes in cell cycle distribution of the cells when being treated with different concentrations of Ro 08-2750 for 24h;the transfection technology was used to study the action of 5 specific Ras binding peptide aptamers on MCF-7 cells,and to detect the proliferation,survival of MCF-7 cells 24h after transfection,as well as active-MAPK and p53 proteins expression and apotosis of MCF-7 cells.Results:Immunofluorescence results showed MCF-7 cells express NGF and its receptor TrkA protein;cell counting kit(CCK-8) showed that Ro 08-2750 can inhibit cell proliferation and survival with the dosage,Western blot showed that the expression of active-MAPK increase while the expression of p53 decreased with the dosage;Flow cytometry results suggest that Ro 08-2750 treated cells appeared apoptotic peak,the content of S-phase cells increased,and G2 phase cells decreased;Fluorescence microscopy showed that transfection efficiency was above 70%in MCF-7 cells,Cell counting kit(CCK-8) showed that cell proliferation and survival after being transfected with pPER39R decreased significantly;Peptide aptamer pPER39R can induce the expression of p53 and significant cell apoptosis;pPER39R have no effect on normal DRG cells.Conclusion:Nerve growth factor signaling pathway exists in human breast cancer cells MCF-7.NGF-specific antagonist Ro 08-2750 can inhibit the growth and survival of breast cancer cell in vitro,and induce apoptosis which might be associated with reduced expression of p53 and increased expression of p-ERK 1/2 protein.Aptamer pPER39R can significantly inhibit the proliferation and survival of MCF-7 cells.Aptamer pPER39R can induce apoptosis of MCF-7 cells.This result may be caused by the increase of the p53.And pPER 39R had no inhibitory effect on normal NGF signaling pathway of DRG cells.It is suggested that pPER 39R has mutation-specific inhibition of Ras and has a larger value in further study.