title: [Construction of has-miR-335 lentiviral vector and verification of the target gene of miR-335].
abstract: OBJECTIVE
  To construct a lentiviral vector of miR-335 gene and verify the target gene of miR-335.
  
  
  METHODS
  The precursor sequence of miR-335 gene was amplified from the genomic DNA by PCR and cloned into the lentiviral vector PLVTHM labeled with GFP. Real-time quantitative RT-PCR was used to detect miR-335 and RASA1 expression in different colorectal cancer cell lines. A recombinant vector psiCHECK-2-RASA1 containing RASA1 3'UTR was constructed followed by site-directed mutagenesis of RASA1 3'UTR to establish the vector psiCHECK-2-RASA1-Mut. Co-transfection of hsa-mir-335 or a NC with these recombined vectors in HEK293A and SW480 cells was performed, and dual-luciferase reporter assay was utilized to examine the changes in luciferase activities. The recombinant PLVTHM-miR335 plasmid was packaged into mature lentivirus by 293FT cells and used to infect SW620 cells. Flow cytometry was employed for sorting the GFP+ cells. The expression of miR-335 and RASA1 were determined by qRT-PCR, and Western blotting was used to detect the expression of RASA1 protein in SW620 cell lines.
  
  
  RESULTS
  The recombinant lentiviral vector PLVTHM-miR335, psiCHECK-2-RASA1 and the mutation expression vector psiCHECK-2-RASA1-Mut were successfully constructed. Dual-luciferase reporter assay showed that miR-335 decreased luciferase activity in cells co-transfected with psiCHECK-2-RASA1. The expression of miR-335 in SW620 cells infected with the lentivirus PLVTHM-miR335 was significantly increased, but the expression of RASA1 showed only slight changes. Overexpression of miR-335 suppressed the expression of RASA1 protein in SW620 cells.
  
  
  CONCLUSIONS
  We have successfully constructed the lentiviral vector containing mir-335 gene and a SW620 cell line with miR-335 overexpression. MiR-335 can suppress RASA1 gene expression by targeting the specific sequence of RASA1 3'UTR.
authors.affiliation: Department of Pathology, College of Basic Medical Sciences, Key Laboratory of Molecule Tumor Pathology of Guangdong Province, Southern Medical University, Guangzhou 510515, China. yh19820818@sina.com
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Yan-Xia
authors.initials: YX
authors.lastname: Lu
authors.forename: Xiao-Jin
authors.initials: XJ
authors.lastname: Wu
authors.forename: Li
authors.initials: L
authors.lastname: Yuan
authors.forename: Chang
authors.initials: C
authors.lastname: Zhou
authors.forename: Chun-Ping
authors.initials: CP
authors.lastname: Zhou
authors.forename: Guo-Bing
authors.initials: GB
authors.lastname: Liu
authors.forename: Xue-Nong
authors.initials: XN
authors.lastname: Li
mesh_terms: D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020729:p120 GTPase Activating Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C556347:MIRN335 microRNA, human; D035683:MicroRNAs; C557248:RASA1 protein, human; D011994:Recombinant Proteins; D020729:p120 GTPase Activating Protein; D049452:Green Fluorescent Proteins