title: [Construction and package of a recombinant adenoviral vector to inhibit miR-21 expression and its effects on HepG2 cells in vitro].
abstract: OBJECTIVE
  To construct the recombinant adenoviral vector which can efficiently inhibit mature miR-21 expression and explore its effects and the underlying mechanisms on hepatoma cell line HepG2.
  
  
  METHODS
  Based on miRNA-sponge technology,  we synthesized 8 duplicated fragments,  fully complementary with the miR-21 sequence and cloned them into a shuttle vector pAdTrack-CMV. The constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria,  then transfected into 293A cells. The recombinant adenovirus was used to challenge HepG2 cells. Mature miR-21 level was detected by real-time PCR. Apoptosis and proliferation of the HepG2 cells were detected by Hochest  33258 staining,  Western blotting and MTT assay.
  
  
  RESULTS
  The restriction enzyme digestion,  DNA sequencing and detection of GFP expression demonstrated that recombinant adenoviral vector was constructed successfully. The recombinant adenovirus inhibited the expression of miR-21 in HepG2 cells,  and also depressed the proliferation of HepG2 cells and promoted the apoptosis.
  
  
  CONCLUSIONS
  The recombinant adenoviral vector we successfully constructed could efficiently reduce the expression of miR-21 in HepG2 cells and depress the proliferation of HepG2 cells.
authors.affiliation: Institute of Laboratory Medicine, General Hospital, Ningxia Medical University, Yinchuan 750004, China. zyl-870331@163.com
authors.forename: Yi-lin
authors.initials: YL
authors.lastname: Zhang
authors.forename: Jian-zhong
authors.initials: JZ
authors.lastname: Tang
authors.forename: Hong-peng
authors.initials: HP
authors.lastname: Liu
authors.forename: Zhao-bo
authors.initials: ZB
authors.lastname: Zhang
authors.forename: Wen-jin
authors.initials: WJ
authors.lastname: Xu
authors.forename: Jun
authors.initials: J
authors.lastname: Wei
authors.forename: Guang-xian
authors.initials: GX
authors.lastname: Xu
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D017209:Apoptosis; D001483:Base Sequence; D049109:Cell Proliferation; D015536:Down-Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D008969:Molecular Sequence Data; D019065:Virus Assembly
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs