H curve of Hep3B cells was shown for each group. The proliferation was assayed in triplicates at 0, 24, 48 and 72 hour post-transfection of siRNAs. (B) Effects of different treatments on the inhibition of cell proliferation were confirmed by the total numbers of colony. (C) Representative images of the CibinetideMedChemExpress ARA290 colony formation assay were shown. Values were given as mean value ?standard deviation (SD) of triplicate. **p < 0.01, *p < 0.05 compared to untreated cell group; #p < 0.05 compared to siRNA cocktail treated cell group.Figures 8 and 9). There was no significant difference in mRNA and protein levels of Cyclin D1, Bcl-2 and Survivin between CONT-siRNA treated cells and untreated ones.Effects of VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail on tube formation in HUVECsA HUVECs angiogenesis model was employed to evaluate the tube formation of HUVECs stimulated by the conditioned medium derived from Hep3B cells transfected with siRNA cocktail, VEGF-siRNA#1, KSP-siRNA#2 and CONT-siRNA. As illustrated in Figure 10, siRNA cocktail or VEGFsiRNA#1 transfected Hep3B cells inhibited HUVECs to form extensive and enclosed tube networks on Matrigel as compared to the CONT-siRNA treated cells and untreated ones (p < 0.05, Figure 10B). However, KSP-siRNA#2 treated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 cells did not affect on tube formation in HUVECs. We also determined the mRNA and protein levels of ANG2 in HUVECs. In normally cultured negative controlcells, the expression of ANG2 mRNA (11.24 ?2.15 ) and protein (18.24 ?1.88 ) was slight, when compared to the untreated cells (Figure 11). CONT-siRNA did not cause any statistical differences compared to untreated cells. In VEGF-siRNA#1 treated cells, the expression of ANG2 mRNA (41.66 ?3.03 , p < 0.05, Figure 11A) and protein (59.62 ?1.84 , p < 0.05, Figure 11B) was significantly reduced compared to untreated cells. siRNA cocktail treated cells (ANG2 mRNA: 39.82 ?2.78 ; protein: 53.86 ?1.84 ) exhibited similar effect with VEGF-siRNA#1 treated cells. In contrast, the result was not reproduced by KSPsiRNA#2, which showed no significant difference in ANG2 expression in HUVECs between KSP-siRNA#2 treated cells and untreated ones (Figure 11).Discussion As tumor cells are characterised by multiple genetic and epigenetic alterations, the single inhibition of one tumourassociated gene as a therapeutic strategy may not beDoan et al. Biological Research 2014, 47:70 http://www.biolres.com/content/47/1/Page 7 ofFigure 5 Effects of different treatments on cell migration in Hep3B cells. The cells with different treatments at 0, 24, 48, and 72 hours. (A) Representative images of the cell migration ability assay were shown. (B) Effects of different treatments on migration ability of Hep3B cells were determined by the cell relative migration distances in different time points. Value were presented as mean value ?standard deviation (SD) of triplicate. **p < 0.01, *p < 0.05 compared to untreated cell group and #p < 0.05 compared to siRNA cocktail treated cell group.sufficient for inhibition of tumor development. It has been well known that gene therapy targeting either VEGF or KSP alone may cause inhibition of HCC growth [14,19]. However, the current finding showed that siRNA cocktail silencing VEGF and KSP together could inhibit the proliferation, migration or invasion of HCC cells better than single siRNA simultaneously. On the other hand, the siRNA cocktail might also increase apoptosis induction in HCC cells. This is a better therapeutic strategy which could.