title: [Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer].
abstract: OBJECTIVE
  To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer.
  
  
  METHODS
  First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM.
  
  
  RESULTS
  It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (P<0.05). The apoptosis rate of A549/Lv-shCyPA (5.01% +/- 0.5%) was higher than control groups (0.35% +/- 0.17%) (P<0.05). Visible tumors were only detectable at 6th day after inoculated by A549/Lv-shCyPA. The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size at 38th days after inoculation compared with the control group (P < 0.05).
  
  
  CONCLUSIONS
  Lentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA, induces the NSCLC cell apoptosis, inhibits the tumor growth. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.
authors.affiliation: Institute of Medicine Henan University of Science and Tochnology, Luoyang 471003, China.
authors.forename: Yan-ming
authors.initials: YM
authors.lastname: FENG
authors.forename: Yi-ming
authors.initials: YM
authors.lastname: WU
authors.forename: Xin-ming
authors.initials: XM
authors.lastname: TU
authors.forename: Zheng-shun
authors.initials: ZS
authors.lastname: XU
authors.forename: Wei-dong
authors.initials: WD
authors.lastname: WU
mesh_terms: D000818:Animals; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D045744:Cell Line, Tumor; D021984:Cyclophilin A / Q000235:genetics*; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics*; D051379:Mice; D034741:RNA, Small Interfering
chemical_list: D034741:RNA, Small Interfering; D021984:Cyclophilin A