Influence of RNA interference-induced RhoC gene silencing on behaviors of human umbilical vein endothelial cells

Abstract

Objective To investigate the effects of RhoC gene silencing by RNA interference (RNAi) on behaviors of human umbilical vein endothelial cells (HUVECs) in vitro.Methods The vector containing shRNA targeting RhoC gene was constructed and transfected into HUVECs by Lipofectamine-2000.The expression of Rhoc mRNA and protein in HUVECs was detected respectively by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Wound-healing assay in vitro and three-dimensional cultures were used to evaluate the migration and angiogenesis capacity of HUVECs.The changes in the adhesion and proliferation of the cells were examined by methyl thiazol tetrazolium (MTT)assay.Results In RhoCshRNA-transfected group and control group,the expression of RhoC mRNA and protein was ( 10.93 ± 4.72 ) ％ and ( 19.95 ± 7.31 ) ％,and ( 56.77 ± 13.79 ) ％ and ( 62.06 ± 22.75 ) ％respectively,and the per-cent wound closure was (45.08 ± 12.93 )％ and (73.49 ± 10.83 )％ respectively.The absorbance (A) value of proliferation was 1.002 ± 0.283 and 1.629 ± 0.192.The number of cordlike was 2.85 ± 1.08 and 4.46 ± 1.38,and the adhesion vasue was 0.795 ± 0.097 and 0.907 ±0.178.The expression of RhoC was suppressed by transfecting RhoCshRNA into HUVEs,meanwhile,the migration,proliferation and angiogenesis capacity of HUVECs were decreased. No significant difference was found in the adhesion of the cells between RhoCshRNA-transfected group and control group.Conclusion RNAi-induced RhoC gene silencing may decrease the capacity of HUVE migration,proliferation and angiogenesis in vitro. Key words: RNA interference; RhoC; Human umbilical vein endothelial cells; Cell migration; Angiogenesis