title: [Construction of has-microRNA-338-3p lentiviral vector and verification of its target gene].
abstract: OBJECTIVE
  To construct a lentiviral expression vector of has-miR-338-3p and verify its target gene.
  
  
  METHODS
  The pre-miR-338-3p was synthesized and inserted into pLV-THM, and the recombinant plasmid pLV-THM-miR-338-3p was confirmed by restriction endonuclease analysis and DNA sequencing. 293T cells were co-transfected with the lentiviral vector pLV-THM-miR-338-3p, psPAX2 and pMD2.G, and the supernatant containing the lentivirus particles was harvested to determine the virus titer and used to infect SW-620 cells. Flow cytometry was employed for sorting the GFP-positive cells. The expression of miR-338-3p was determined using real-time RT-PCR and the expression of SMO protein was detected with Western blotting in the infected SW-620 cells. The invasiveness of the infected SW-620 cells was assessed using Transwell assay.
  
  
  RESULTS
  Restriction enzyme digestion and DNA sequencing demonstrated successful construction of the lentiviral vector pLV-THM-miR-338-3p. SW-620 cells infected with pLV-THM-miR-338-3p showed a significantly increased expression of miR-338-3p, and the overexpression of miR-338-3p suppressed the expression of SMO protein and the invasiveness of the cells.
  
  
  CONCLUSIONS
  The successful construction of the lentiviral vector pLV-THM-miR-338-3p and the establishment of a SW-620 cell line with miR-338-3p overexpression provide the basis for further study of the molecular function of miR-338-3p in colorectal carcinoma. MiR-338-3p can suppress SMO gene expression to inhibit the invasiveness of colorectal carcinoma cells.
authors.affiliation: Department of General Surgery, Southern Medical University, Guangzhou, China. navyd999@tom.com
authors.forename: Haijun
authors.initials: H
authors.lastname: Deng
authors.forename: Kai
authors.initials: K
authors.lastname: Sun
authors.forename: Chen
authors.initials: C
authors.lastname: Guo
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Guoxin
authors.initials: G
authors.lastname: Li
mesh_terms: D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*
chemical_list: C532050:MIRN338 microRNA, human; D035683:MicroRNAs; D049452:Green Fluorescent Proteins