title: Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure.
abstract: OBJECTIVE
  To construct a lentivirus-based inhibitor with specific secondary structure that could exert long-term suppression on microRNA-338-3p (miR-338-3p), thus elucidating its molecular function in colorectal carcinoma cells.
  
  
  METHODS
  The miR-338-3p inhibitor sequence was synthesized and inserted into pLV-THM plasmid. HEK-293T cells were co-transfected with the lentiviral vectors pLV-THM-miR-338-3p-inhibitor, psPAX2, and pMD2.G. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and then used to infect colorectal carcinoma-derived SW-620 cells. eGFP(+) cells were sorted using flow cytometry. The expression of miR-338-3p in SW-620 cells was determined with real-time RT-PCR, and the expression of the smoothened (SMO) protein was detected using Western blot analysis. The migration ability of the transfected SW-620 cells was assessed with transwell assay.
  
  
  RESULTS
  Restriction endonuclease analysis and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p-inhibitor was successfully constructed. The expression of miR-338-3p in SW-620 cells was significantly decreased by infection with the lentivirus pLV-THM-miR-338-3p-inhibitor. Moreover, the down-regulated expression of miR-338-3p caused up-regulated expression of the SMO protein in SW-620 cells, which showed significantly enhanced migration in transwell assay.
  
  
  CONCLUSIONS
  The construction of the lentiviral vector pLV-THM-miR-338-3p-inhibitor with specific secondary structure provides a basis for further studies the molecular function of miR-338-3p in colorectal carcinoma. miR-338-3p may suppress SMO gene expression and thereby inhibit colorectal carcinoma migration.
authors.affiliation: Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China. sunkai9602@sina.com
authors.forename: Kai
authors.initials: K
authors.lastname: Sun
authors.forename: Chen
authors.initials: C
authors.lastname: Guo
authors.forename: Hai-jun
authors.initials: HJ
authors.lastname: Deng
authors.forename: Jing-qing
authors.initials: JQ
authors.lastname: Dong
authors.forename: Shang-tong
authors.initials: ST
authors.lastname: Lei
authors.forename: Guo-xin
authors.initials: GX
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D003106:Colon / Q000166:cytology / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000235:genetics*; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012007:Rectum / Q000166:cytology / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: C532050:MIRN338 microRNA, human; D035683:MicroRNAs