title: [Construction of human Bcl-6 3'UTR reporter vector and expression vector and their functional assessment].
abstract: OBJECTIVE
  To observe the direct regulation of miR-127 on Bcl-6 and the effect of Bcl-6 in rescuing miR-127-induced cell cycle and cell growth inhibition.
  
  
  METHODS
  The 3'UTR and coding region of human bcl-6 gene were amplified by PCR and cloned into pcDNA3.0-Luc and pcDNA3.0-Flag vectors, respectively. Mutations were introduced into the seed sequences of the predicted miR-127 target sites within the Bcl-6 3'UTR using recombinant PCR. Luciferase assay was used to verify the direct targeted regulation of miR-127 on Bcl-6. In HepG2 cell models with overexpression or knockdown of miR-12, the changes of cell cycle and cell growth were investigated after transfection with the constructed vectors.
  
  
  RESULTS
  The recombinant plasmids were successfully obtained as confirmed by double digestion and sequence identification. Luciferase assay showed that in 293T and HepG2 cells, miR-127 inhibited the activation of wild-type Bcl-6 3'UTR reporter vector but not mutated Bcl-6 3'UTR vector. Overexpression of miR-127 induced cell cycle arrest at G(2)/M phase and suppressed the growth of HepG2 cells, and these effects were reversed by Bcl-6 overexpression.
  
  
  CONCLUSIONS
  We successfully cloned wild-type and mutated 3'UTR reporter vectors and expression vector of bcl-6 gene and confirmed their biological functions.
authors.affiliation: 1Department of Endocrinology, General Hospital of PLA, Beijing 100853, China; 2Department of Endocrinology, 264 Hospital of Beijing Military Command, Taiyuan 030000, Chin. E-mail: bzy229@sina.cn.
authors.forename: Bai-Yu
authors.initials: BY
authors.lastname: Han
authors.forename: Han-Zhi
authors.initials: HZ
authors.lastname: Cui
authors.forename: Xiang
authors.initials: X
authors.lastname: Yan
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.forename: Hua-Long
authors.initials: HL
authors.lastname: Huang
authors.forename: Zhong-Yi
authors.initials: ZY
authors.lastname: Fan
authors.forename: Jing-Tao
authors.initials: JT
authors.lastname: Dou
mesh_terms: D020413:3' Untranslated Regions; D002453:Cell Cycle; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D008156:Luciferases; D035683:MicroRNAs / Q000235:genetics*; D051560:Proto-Oncogene Proteins c-bcl-6; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; C084103:BCL6 protein, human; D004268:DNA-Binding Proteins; C547276:MIRN127 microRNA, human; D035683:MicroRNAs; D051560:Proto-Oncogene Proteins c-bcl-6; D008156:Luciferases