title: [Construction of P2Y6 constitutive knock down breast cancer cell line and evaluation of its proliferation].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: East China Normal University, Shanghai, China.
authors.forename: Lin-li
authors.initials: LL
authors.lastname: Yang
authors.forename: Rui-mei
authors.initials: RM
authors.lastname: Li
authors.forename: Xin-hua
authors.initials: XH
authors.lastname: Pan
authors.forename: Min
authors.initials: M
authors.lastname: Qian
authors.forename: Bing
authors.initials: B
authors.lastname: Du
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D006801:Humans; D051379:Mice; D034622:RNA Interference; D018048:Receptors, Purinergic P2 / Q000235:genetics*
chemical_list: D018048:Receptors, Purinergic P2; C099845:purinoceptor P2Y6