title: [shRNAs targeting high mobility group box-1 lead to inhibition of E-selectin expression via homeobox A9 in human umbilical vein endothelial cells].
abstract: OBJECTIVE
  To approach the regulatory mechanism of high mobility group box-1 ( HMGB1 ) on the expression of E-selectin in human umbilical vein endothelial cell ( HUVEC ).
  
  
  METHODS
  Homeobox A9 ( HOXA9 ) siRNA was transfected to HUVEC at logarithmic phase, real-time fluorescence quantitative polymerase chain reaction ( real-time qPCR ) and Western Blot were used to determine the HOXA9 mRNA expression and protein expressions; a blank control group and a nonsilence negative control group were set. HUVEC stable transfected with pRNA-u6.1/Neo-HMGB1 shRNA plasmids ( HUVEC with low-expression HMGB1 ) was obtained, and HOXA9 and E-selectin mRNA expressions were determined with real-time qPCR; a nonsilence transfection group served as the negative control. The HOXA9 siRNA was transfected to HUVEC with low-expression HMGB1 as co-transfection group, and the E-selectin expressions was determined with real-time qPCR; a HMGB1 shRNA group and a HOXA9 nonsilence group served as control.
  
  
  RESULTS
  (1) HOXA9 mRNA ( 2(-Δ ΔCT) ) and protein expression ( integral A value ) in blank control group were 1.094±0.115 and 1.031±0.060. Compared with nonsilence transfection group, HOXA9 siRNA transfection group could significantly reduced mRNA and protein expression of HOXA9 [ HOXA9 mRNA ( 2(-Δ ΔCT) ): 0.257±0.030 vs. 1.035±0.091, t = 14.010, P = 0.002; HOXA9 protein ( integral A value ): 0.278±0.042 vs. 0.975±0.014, t = 27.310, P = 0.002 ]. (2) Compared with nonsilence transfection group, HMGB1 shRNA transfection could up-regulate HOXA9 mRNA expression in HUVEC ( 2(-Δ ΔCT) : 2.519±0.278 vs. 0.856±0.063, t = 10.100, P = 0.001 ), also could down-regulate E-selectin mRNA expression ( 0.311±0.046 vs. 1.080±0.201, t = 7.415, P = 0.000 ). (3) Compared with HOXA9 nonsilence group and HMGB1 shRNA group, HMGB1 shRNA and HOXA9 siRNA co-transfected HUVEC cells could significantly elevate E-selectin mRNA expression ( 2(-Δ ΔCT) : 3.445±0.428 vs. 1.085±0.212, 1.004±0.104, t(1) = 8.507, t(2) = 9.603, both P < 0.001 ).
  
  
  CONCLUSIONS
  HMGB1 may regulate E-selectin expression through the HOXA9 regulation in HUVEC.
authors.affiliation: Department of Critical Care Medicine, the First Hospital, China Medical University, Shenyang 110001, Liaoning, China. Corresponding author: Ma Xiaochun, Email: xcma2972@sina.com.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Zhang
authors.forename: Lili
authors.initials: L
authors.lastname: Jiao
authors.forename: Zhenggang
authors.initials: Z
authors.lastname: Luan
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Ma
mesh_terms: D015536:Down-Regulation; D019040:E-Selectin / Q000235:genetics*; D024243:HMGB1 Protein / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology*; D006801:Humans; D012333:RNA, Messenger; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
chemical_list: D019040:E-Selectin; D024243:HMGB1 Protein; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C546936:SELE protein, human; C097899:homeobox protein HOXA9