ShRNA targeting human TGF-β1 gene suppresses TGF-β1 gene over expression in human umbilical vein endothelial cell line after anoxia/reoxygenation injury

Abstract

Objective To construct short hairpin RNA (shRNA) eukaryotic expression vector targeting human TGF-β1 gene,and to evaluate the effect of this shRNA expression vector on human umbili-cal vein endothelial cells (HUVEC) after anoxia/reoxygenation (A/R) injury. Methods The eukaryotic expression vector targeting TGF-β1 mRNA was designed, synthesized and constructed, and the HUVEC were transfected by well-done shRNA expression vector firstly,and then underwent the A/R experiments. The semi-quantification reverse transcriptive PCR(RT-PCR)was performed to detect the expression of TGF-β1 mRNA,and immunohistochemistry was used to detect the expression of TGF-β1 protein at 12th h after A/R experiments. Results The inhibition rate of the well-done shRNA expression vector was(46.4 ±3.7 )%. The absorbance (A) values of the anoxia-reoxygenation groups T, H, L and C were 0. 252 ± 0.032,0. 385 ± 0.037,0. 406 ± 0.057 and 0. 419 ± 0. 04 respectively, which were higher than that of the non A/R group N (0. 103 ± 0. 008, P < 0. 01 ). Among the A/R groups, the A value of group T transfected with pT12 was significantly lower than that of the other groups. The overexpression of TGF-β1 mRNA caused by A/R injury was also downregulated in group T (P < 0.05 ). Conclusion The shRNA eukaryotic expression vectors targeting human TGF-β1 gene had significant inhibitory effects on the overex- pression of TGF-β1 mRNA and protein caused by A/R injury in HUVEC. Key words: Anoxia/reoxygenation; Umbilical veins endothelial cell; Transforming growthfactor-beta-1 ; shRNA