Xpression. This LLY-507 solubility observation was also demonstrated by angiogenesis and ANG2 expression on the HUVECs induced by Hep3B cell culture media following siRNAs treatment. A previous study indicatedthat HUVECs was induced to form new blood vessels with higher VEGF concentration [31]. In addition, ANG2 which is expressed in the areas undergoing vascular remodeling and leads to decreased vessel maturation and enhanced vessel sprouting could be upregulated by VEGF in endothelial cells [32]. ANG2 inhibition prevents the growth of new vessels by endothelial sprout formation [33]. In our study, the results showed that inhibition of capillary tube-like structure formation and suppression of ANG2 expression in HUVECs by siRNA cocktail was relatively equal when compared to VEGF-siRNA. Meanwhile, KSP-siRNA showed no effect on inhibiting tube formation as well as ANG2 expression in HUVECs. These results demonstrated that VEGF acts as a KSP upstream regulator promoting the effect of KSP on Hep3B cells. More importantly, our estimates were strongly supported by another study reporting a strong upregulation of KSP or Eg5 expression after application of recombinant human VEGF on the differentiated day-13 chick chorio-allantoic membrane (CAM). The results showed that numerous known genes encoding mitotic PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26104484 kinesins were consistently upregulated by VEGF, including KIF4A, KIF11/Eg5, KIF15, KIF20A/Mklp2 and KIF23 [34].Conclusion This study showed that the expression of KSP was inhibited by VEGF-siRNA, suggesting that VEGF serves as anDoan et al. Biological Research 2014, 47:70 http://www.biolres.com/content/47/1/Page 12 ofFigure 11 Effects of different treatments on ANG2 expression in HUVECs cultured with Hep3B supernatant. HUVEC complete medium (without VEGF) was used as a negative control. (A) ANG2 mRNA expression levels were showed by Real-time qRT-PCR analysis. (B) ANG2 protein in HUVECs was determined by Western Blot analysis. (C) Densitometric analysis of ANG2 protein was made relative to -actin. Values were given as mean value ?standard deviation (SD) of triplicate. *p < 0.05 compared to untreated cell group.upstream regulator of KSP gene expression. Although there were several reports using siRNA to inhibit VEGF or KSP, combined siRNA therapy to simultaneously reduce VEGF and KSP expression was proved to be an effective approach to inhibit cell growth and induce apoptosis of HCC cells. This could be a new targeted strategy to eradicate HCC cells.Methods This experimental study was approved by the Committee for Ethics in Research, University of Science, Vietnam National University.Culture of cellsCRL-1730) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Hep3B cells were thawed and cultured in DMEM-F12, supplemented with 10 FBS and 0.5 antibiotic-mycotic (All were bought from Sigma-Aldrich, St. Louis, MO, USA). HUVECs cells were thawed and cultured in endothelial cell growth medium-2 (EGM-2) (Lonza, Walkersville, MD, USA), supplemented with 10 FBS and 0.5 antibioticmycotic. All cells were maintained at 37 , 5 CO2.Transfection of siRNAsHep3B cell line (hepatocellular carcinoma cells, HB-8064), HUVECs cell line (human umbilical vein endothelial cells,The sequences of the siRNAs targeting VEGF-A (VEGFA-siRNA, also simply referred to as VEGF-siRNA), siRNAs targeting KSP (KSP-siRNA) and mismatched siRNA (CONT-siRNA) were shown in Table 1. All siRNAs were synthesized by Bioneer (Daejeon, Republic of Korea). EachDoan et al.