title: [Effects of core-binding factor α1 gene silenced by siRNA on calcification of vascular smooth muscle cells induced by high phosphate].
abstract: OBJECTIVE
  To explore the effects of core-binding factor α1 (Cbfα-1) gene silenced by siRNA on osteogenic differentiation and calcification of vascular smooth muscle cells (VSMC) induced by high phosphate in vitro.
  
  
  METHODS
  VSMC were cultured in vitro and passaged 3 to 8 times. Four pairs of Cbfα-1 siRNA were designed and synthesized. Transfection was performed with cationic lipid vectors (Lipofectamine 2000). Transfection conditions were optimized by the FAM fluorescent labeling-siRNA to screen effective siRNA sequences by reverse transcription-polymerase chain reaction (RT-PCR). After transfection with effective siRNA sequences, VSMCs were divided into 4 groups: (1) normal phosphate (Pi 1.3 mmol/L); (2) high phosphate (Pi 2.6 mmol/L); (3) siRNA transfection: high phosphate+Cbfα-1-siRNA; (4) negative transfection control: high phosphate+negative control siRNA. Cbfα-1 and osteopontin (OPN) mRNA and protein expression were detected by RT-PCR and Western blotting. Calcium deposition was visualized by Alizarin stain method.
  
  
  RESULTS
  The transfection efficiency was around 55% with a concentration of Cbfα-1 siRNA 100 nmol/L and Lipo 8 µl/ well. Cbfα-1 siRNA 1952 was chosen as the effective sequence with a suppression ratio up to 81.8%. At 24 and 48 h post-transfection, the expression of Cbfα-1 mRNA was significantly lower in siRNA transfection group than that in high phosphate group (0.335 ± 0.059 vs 0.714 ± 0.106, 0.574 ± 0.036 vs 0.726 ± 0.086, all P < 0.01) . At 48 and 72 h post-transfection, the expression of Cbfα-1 protein in siRNA transfection group was significantly lower than that in high phosphate group (both P < 0.01) . While Cbfα-1 gene was silenced by siRNA in siRNA transfection group, the mRNA and protein expression of OPN significantly declined (all P < 0.05) and calcium deposition in cell layers decreased.
  
  
  CONCLUSIONS
  Cbfα-1 siRNA can effectively inhibit the expression of Cbfα-1 mRNA and protein in VSMC and thus suppress the transformation of VSMC into osteoblast-like cells and calcification induced by high phosphate. Cbfα-1 may become a potential therapeutic target in vascular calcification of chronic kidney disease.
authors.affiliation: Department of Blood Purification, Fuzhou General Hospital of Nanjing Command, Fuzhou 350025, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Blood Purification, Fuzhou General Hospital of Nanjing Command, Fuzhou 350025, China.
authors.forename: Peidan
authors.initials: P
authors.lastname: Wei
mesh_terms: D000818:Animals; D002114:Calcinosis; D002454:Cell Differentiation; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics*; D020868:Gene Silencing; D032342:Muscle Cells / Q000166:cytology / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D010012:Osteogenesis / Q000187:drug effects*; D010710:Phosphates / Q000009:adverse effects; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D010710:Phosphates; D012333:RNA, Messenger; D034741:RNA, Small Interfering