title: [Construction of an oncolytic adenovirus expressing small hairpin RNA and targeting the SATB1 gene].
abstract: OBJECTIVE
  To construct an oncolytic adenovirus with the DD3 promoter regulation, expressing small hairpin RNA and targeting the SATB1 gene (SATBI-shRNA), and to evaluate its potential for inhibiting the growth of human prostatic carcinoma cells (LNCaP) in vitro.
  
  
  METHODS
  SATB1-shRNA expression cassettes containing the H1 promoter were produced by PCR from pSilencer3. 1-SATB1 and inserted into the pZD55 vector, and the recombinant plasmid pZD55-SATB1-shRNA was constructed, pZD55SATB1-shRNA and pZXC2-DD3-E1A were digested with EcoRV and Xba I , and the obtained expression cassettes linked each other to construct the recombinant plasmid pDD3-ZD55-SATB1, which was cotransfected with the pBHGE3 packaging plasmids mixture into 293 cells by Effectence. The recombined adenoviruses DD3-ZD55-SATB1 were identified by PCR. Viruses were propagated on HEK293 cells and purified by standard techniques, and the functional PFU titers determined by plaque assay on 293 cells. The antitumor potential of DD3-ZD55-SATB1 to LNCaP was evaluated by the crystal violet dye method. The expression level of the E1A gene was detected by Western blot, and that of the SATB1 gene in the adenovirus-infected LNCaP cells by both Western blot and RT-PCR.
  
  
  RESULTS
  An oncolytic adenovirus expressing SATB1-shRNA with the DD3 promoter regulation, DD3-ZD55-SATB1, was constructed successfully, whose functional PFU titer was 1.2 x 10(10) PFU/ml. DD3-ZD55-SATB1 showed an obvious cytopathic effect and a selective expression of E1A in the adenovirus-infected LNCaP cells; it exhibited a high LNCaP-targetability and specific SATB1-silencing effect.
  
  
  CONCLUSIONS
  The successful construction of the oncolytic adenovirus DD3-ZD55-SATB1 offers a new tool for researches on the gene therapy for human prostate cancer.
authors.affiliation: Department of Urology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, China.
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Mao
authors.forename: Jun-Nian
authors.initials: JN
authors.lastname: Zheng
authors.forename: Wang
authors.initials: W
authors.lastname: Li
authors.forename: Jun-Qi
authors.initials: JQ
authors.lastname: Wang
authors.forename: Jia-Cun
authors.initials: JC
authors.lastname: Chen
authors.forename: Xiao-Qing
authors.initials: XQ
authors.lastname: Sun
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002277:Carcinoma / Q000628:therapy; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D036961:Matrix Attachment Region Binding Proteins / Q000235:genetics*; D050130:Oncolytic Virotherapy / Q000379:methods; D050504:Oncolytic Viruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000628:therapy; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D036961:Matrix Attachment Region Binding Proteins; D034741:RNA, Small Interfering; C076189:SATB1 protein, human