shRNAs targeting high mobility group box-1 lead to inhibition of E-selectin expression via homeobox A9 in human umbilical vein endothelial cells
10.3760/cma.j.issn.2095-4352.2015.08.009
- VernacularTitle:短发夹高迁移率族蛋白B1真核细胞表达载体通过HOXA9下调人脐静脉血管内皮细胞表达E-选择素
- Author:
Xiaojuan ZHANG
;
Lili JIAO
;
Zhenggang LUAN
;
Xiaochun MA
- Publication Type:Journal Article
- Keywords:
Sepsis;
Vascular endothelial cell;
High mobility group protein;
Homeobox gene;
E-selectin;
Transfection;
Eukaryotic expression vector
- From:
Chinese Critical Care Medicine
2015;(8):662-666
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo 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.Results① HOXA9 mRNA (2-ΔΔCT) and protein expression (integralA 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-ΔΔCT): 0.257±0.030 vs. 1.035±0.091,t = 14.010,P = 0.002; HOXA9 protein (integralA 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-ΔΔCT: 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-ΔΔCT: 3.445±0.428 vs. 1.085±0.212, 1.004±0.104,t1 = 8.507, t2 = 9.603, bothP< 0.001).Conclusion HMGB1 may regulate E-selectin expression through the HOXA9 regulation in HUVEC.
