title: [Expression of miR-145 in transfected HeLa cells and its effect on the targeted inhibition of CDK6].
abstract: OBJECTIVE
  To construct the eukaryotic expression vector of miR -145 and evaluate the inhibitory effect of miR-145 targeting cyclin-dependent kinase 6 (CDK6) gene on the proliferation of human cervical carcinoma cells.
  
  
  METHODS
  The gene sequence of miR-145 was synthesized and cloned into pcDNA(TM);6.2-GW to construct recombinant plasmid pcDNA(TM);6.2-GW-miR-145. HeLa cells were divided into miR-145 group (transfected with the recombinant plasmid), NC group (untransfected) and blank group (transfected with a blank plasmid). The transcription levels of miR-145 and CDK6 were detected using real-time quantitative PCR (qRT-PCR), and Western blotting was used to evaluate the CDK6 protein expression. In addition, the inhibitory effect of miR-145 targeting CDK6 on the proliferation of HeLa cells was measured by MTT assay.
  
  
  RESULTS
  Sequencing and reverse transcrip0tion PCR demonstrated that we constructed successfully the recombinant plasmid pcDNA(TM);6.2-GW-miR-145, and that the miR-145 expression level in miR-145 group was significantly higher than that in blank group. The qRT-PCR and Western blotting showed that the CDK6 expression level in miR-145 group was significantly lower than that in blank group. Furthermore, miR-145 significantly inhibited the proliferation of HeLa cells (P<0.05).
  
  
  CONCLUSIONS
  The eukaryotic expression vector carrying miR-145 was successfully constructed, and miR-145 over-expression in HeLa cells could reduce the expression of CDK6.
authors.affiliation: Medical College, Yan'an University, Yan'an 716000, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Manpeng
authors.initials: M
authors.lastname: Huo
authors.forename: Mingtao
authors.initials: M
authors.lastname: Mu
authors.forename: Junjun
authors.initials: J
authors.lastname: Liu
authors.forename: Jiming
authors.initials: J
authors.lastname: Han
mesh_terms: D049109:Cell Proliferation; D051361:Cyclin-Dependent Kinase 6 / Q000037:antagonists & inhibitors* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000502:physiology; D014162:Transfection
chemical_list: C541010:MIRN145 microRNA, human; D035683:MicroRNAs; D051361:Cyclin-Dependent Kinase 6