[Construction of P2Y6 constitutive knock down breast cancer cell line and evaluation of its proliferation].

To construct the constitutive P2Y6 knock down breast cancer cell line with shRNA technology and provide a basis for discovering how P2Y6 regulates tumorigenesis and progression in breast cancer. The paired oligo nucleotides targeting human P2Y6 gene were synthesized and annealed into linear PLKO lentiviral vector digested by EcoRI and AgeI. The recombined vector which was identified by double digest with EcoRI and NdeI and DNA sequencing was packaged in 293T cells together with psPAX2 and pMD2.G. Virus in culture supernatant was concentrated, three recombinant vectors were transfected into BT549 cells, and the constitutive P2Y6 knock down cells were selected by puromycin. The efficiency of RNA interference was detected by RT-PCR and Western blotting. MTS assay was used to detect the influence of P2Y6 on breast cancer cells'proliferation. The inserted sequence was proven to be correct by DNA sequencing. After stable transfection of P2Y6 shRNA, the expression of P2Y6 in BT549 cells was decreased obviously in both protein and mRNA level. But no obvious influence on proliferation was found in P2Y6 gene knock down cells. The constitutive P2Y6 knock down cell line was successfully constructed in BT549 cells. Whereas, no obvious correlation was found between the expression of P2Y6 and breast cancer cell proliferation.