RNA Interference Knock Down CCR7Expression Ability To Inhibit Breast Cancer Cell Migration

Objective:1)、detecting CCR7protein expression in various breast cancer cell lines;2)、build CCR7-shRNA expression vector and silence effect on the high CCR7expression of breast cancer cells.3)、detecting CCR7effects on breast cancer cell migration ability.Methods:1)、collect a variety of breast cancer cells, for example, MDA-MB-231、4T1、MCF10A、HCC1937、MDA-MB-361、HCC1580、HCC1806、184A1、184B5、 MCF10A、MDA-MB-468、MCF-7,detecting CCR7protein expression level of breast cancer cells by Western blotting test mentioned.2)、design and build target CCR7gene short hairpin RNA eukaryotic expression plasmid, identification the success of CCR7-shRNA expression vector to construct after double enzyme digestion with BamHI enzymes/EcoRI enzymes.and transfection the high CCR7expression of breast cancer cells which tested by Western blotting, and respectively named A#, B#,C#,G#,H#,then select CCR7expression-shRNA stability breast cancer cell lines by puromycin. Finally detecting interference effect by Western blotting,and further optimize the transfection conditions, to achieve the best effect of interference; Finally select a kind of breast cancer cells with the best interference effect which transfected by shRNA eukaryotic expression plasmid is article, which was CCR7expression-shRNA stably breast cancer cell lines.3), detecting migration ability of the breast cancer cell lines which silence CCR7stably through cell scratch test.Results:1),Western blotting test results show that CCR7protein have expression in6kinds of breast cancer cells, which the strongest expression quantity in MDA-MB-231cells and4T1cells, the stronger expression quantity in T47D cells, MDA-MB-468cells,HCC1937cells, the weakest in MCF-7cell.HCC1580cells,HCC1806cells,184A1cells,184B5cells,MCF10A cells, MDA-MB-361cells did not test expression quantity clearly.Finally,we select the MDA-MB-231breast cancer cells for subsequent experiments.2),the enzyme identification proved that target CCR7gene short hairpin RNA eukaryotic expression plasmid was successfully by designed and builded, then we transfect MDA-MB-231breast cancer cells, in the end detecting MDA-MB-231breast cancer cells after CCR7silence effect by Western blotting, the results show CCR7-shRNA G#can effectively inhibit endogenous CCR7protein expression in MDA-MB-231cells.3), the results of cell scratch experiment show that the control ShLacz scratches the healing degree of MDA-MB-231cells reached70%, while in the experimental group CCR7-shRNA G#cell healing degree is46%.Conclusion:1), chemokine receptors CCR7have a variety of high expression in breast cancer cell lines, especially in MDA-MB-231cells, point out CCR7expression level may be some correlation with the incidence of breast cancer metastasis, breast cancer cell line MDA-MB-231can be used as a research object about CCR7follow-up related experiments.2), CCR7gene targeting shRNA expression vector to construct is correct, transfect MDA-MB-231and get CCR7expression-shRNA cell lines stably, which laid a preliminary foundation in further discuss chemokine receptors CCR7in breast cancer occurrence, development process correlation.3), MDA-MB-231breast cancer cells after knock down CCR7expression inhibits cell migration ability.