He proliferation, migration, invasion and induce apoptosis of Hep3B cells was better than each siRNA alone. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin following the treatment of siRNA cocktail as compared to single siRNA simultaneously. Our results corresponded with several previous studies reporting the influences of siRNA cocktail on cell growth and apoptosis of gastric cancer cells [28], pancreatic cancer cells [29] and colorectal cancer cells [30]. The siRNA cocktail exhibited specific and high efficiency on silencing multi genes simultaneously and would have great potential for therapeutic siRNA applications.Doan et al. Biological Research 2014, 47:70 http://www.biolres.com/content/47/1/Page 11 ofFigure 10 The tube formation inhibition of different treatments was detected in HUVECs angiogenesis model. (A) Representative photographs of each treatments were shown. (B) The total numbers of branching points were decreased by treatment of siRNA cocktail, VEGF-siRNA#1 and KSP-siRNA#2 compared to the untreated. Values were given as mean value ?standard deviation (SD) of triplicate. *p < 0.05 compared to untreated cell group.Interestingly, we found that VEGF-siRNA exhibited significant inhibition on PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 KSP mRNA and protein levels, while the KSP-siRNA alone did not show any effect on VEGF expression. We assumed that VEGF might be acted as a KSP upstream regulator, which could probably lead to downregulation of KSP expression. The fact that VEGFsiRNA presented similar KSP-siRNA effects on proliferation, migration, invasion and apoptosis of Hep3B cells suggesting that one of inhibiting tumor growth mechanisms of VEGF-siRNA functioned through inhibition of KSP expression. Furthermore, we also found that the AZD-8835 web expression of KSP protein was suppressed to a greater extent in the siRNA cocktail treated group than that in the KSP-siRNA or VEGF-siRNA alone group, indicating a significant silencing effect of siRNA cocktail on KSP protein expression. The results were supported by the highest inhibition of proliferation, migration, invasion and induction of apoptosis on Hep3B cells by the siRNA cocktail as compared to each of siRNAs alone. In contrast, suppression on VEGF expression in Hep3B cells by the siRNA cocktail was similar to that by the VEGF-siRNA, which supported the observation that KSP-siRNA did not influence VEGF protein expression. This 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 upregu.