title: Geminin interference facilitates vascular smooth muscle cell proliferation by upregulation of CDK-1.
abstract: OBJECTIVE
  Geminin has been correlated with vascular smooth muscle cell (VSMC) proliferation, but its mechanism is unclear. We selectively silenced the geminin gene of rat VSMCs by using RNAi technology and examined how geminin regulated VSMC proliferation.
  
  
  METHODS
  By using RNA interference in A10 cells and flow cytometry, (3)H-thymidine and 5-ethynyl-2'-deoxyuridine (EdU) measurements were used to detect VSMC proliferation. We performed a Western blot, polymerase chain reaction, and immunohistochemistry to detect the expression and location of geminin and cyclin-dependent kinase-1 (CDK1) in VSMCs.
  
  
  RESULTS
  Silencing geminin significantly increased (3)H-thymidine and EdU incorporation in VSMCs. We observed a significant increase in (3)H-thymidine incorporation 24 h after a serum challenge in the geminin-RNAi-lentiviral vector group (4401.38 ± 438.39 cpm/mg), versus the non-targeting geminin-lentiviral vector (2836.88 ± 476.18 cpm/mg) and control groups (3069.50 ± 508.18 cpm/mg; P < 0.05). In the geminin-RNAi-lentiviral vector group, the EdU-positive cell rate was significantly increased (0.75 ± 0.03; P < 0.05), versus the non-targeting geminin-lentiviral vector (0.41 ± 0.0) or control group (0.40 ± 0.03). Geminin promoted VSMC proliferation, accelerating G0/G1-S cell-cycle progression (G0/G1 cells, 10 % decrease; S-phase cells, approximate 6 % increase) 12 h after serum withdrawal. Both CDK1 protein and mRNA expression were significantly increased in the positive group versus the controls. The immunofluorescence and co-immunoprecipitation results revealed a close interaction existed between CDK1 and the geminin gene in VSMC proliferation.
  
  
  CONCLUSIONS
  Geminin gene inhibition could augment VSMC proliferation by increasing CDK1 expression; thus, geminin may be a potential target for treating vascular diseases, specifically VSMCs.
authors.affiliation: Department of Cardiology, Southwest Hospital, Third Military Medical University, Gaotanyan St, Shapingba District, Chongqing, 400038, People's Republic of China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Zhouqin
authors.initials: Z
authors.lastname: Jiang
authors.forename: Linyu
authors.initials: L
authors.lastname: Li
authors.forename: Yeping
authors.initials: Y
authors.lastname: Zhou
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Song
authors.forename: Maoqin
authors.initials: M
authors.lastname: Shu
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis*; D064248:Geminin / Q000172:deficiency* / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D064248:Geminin; D034741:RNA, Small Interfering; D018844:Cyclin-Dependent Kinases