The siRNA cocktail targeting VEGF and HER2 inhibition on the proliferation and induced apoptosis of gastric cancer cell

Abstract

The aim of this study was to investigate the inhibitory effect of a siRNA cocktail targeting Vascular endothelial growth factor (VEGF) and Human epidermal growth factor receptor 2 (HER2) on cell proliferation, induced apoptosis and the expression of VEGF and HER2 in human gastric carcinoma cell. The silencing rate of pre-designed siRNAs that targeted VEGF and HER2 was detected by Real-time Quantitative PCR (RT-QPCR) analysis. Furthermore, the best silencing siRNA that targeted VEGF and HER2 was prepared as a cocktail to co-knockdown VEGF and HER2 expression at both mRNA and protein levels which were detected by RT-QPCR and Western blot analysis. Cell proliferation inhibition rates were determined by CCK8 assay. The effect of siRNA cocktail on cell apoptosis was determined by flow cytometry. The migration inhibition of siRNA cocktail was analyzed by wound-healing assay. The ability of VEGF to induce endothelial cells to proliferate was examined in HUVECs by the method of tube formation assay. The pre-designed siRNAs could inhibit VEGF and HER2 mRNA level. siRNA cocktail, and co-downregulation of VEGF and HER2 result in significant inhibition of gastric cancer growth and migration in vitro. The inhibition of VEGF and HER2 expressions can induce apoptosis of SGC-7901 cells.

Figures

Fig. 1

The mRNA relative level of VEGF (a) and HER2 (b) with pre-designed siRNAs treatments in SGC-7901 cells by RT-QPCR. Values were given as mean ± SD of three separate experiments with triplicate wells per condition. *P < 0.01 compared with untreated and NC_siR

Fig. 2

The mRNA relative level of VEGF (a) and HER2 (b) with different treatments in SGC-7901 cells by RT-QPCR. Values were given as mean ± SD of three separate experiments with triplicate wells per condition. *P < 0.01 compared with untreated, #P < 0.05 compared with untreated

Fig. 3

The protein relative level of VEGF and HER2 with different treatments in SGC-7901 cells by Western blot. Values were given as mean ± SD of three separate experiments with triplicate wells per condition. *P < 0.05 VEGF protein relative level compared with untreated and NC_siR, #P < 0.05 HER2 protein relative level compared with untreated and NC_siR

Fig. 4

Cell proliferation was analyzed by CCK8 assay. Growth curve of SGC-7901 cells was shown for each treatment at 0, 24, 48, 72, and 96 h. *P < 0.01 compared with untreated, #P < 0.05 compared with untreated

Fig. 5

Cell migration inhibition effects of targeting VEGF and/or HER2 on SGC-7901. a The cells with different treatments at 0, 24, 48, and 72 h (100×). b Cell relative migration distances in different time point treatments were presented as mean ± SD. *p < 0.01, #p < 0.05 compared with untreated and NC_siR

Fig. 6

Cell apoptosis was detected by Annexin V-FITC/PI double staining and FCM analysis. a Apoptosis rates were measured by FCM analysis after AnnexinV/PI staining. b Percentage of dual-positive (Annexin V and PI were positive) cells from three independent experiments was quantified and presented as mean ± SD. *p < 0.01 compared with untreated and NC_siR, #p < 0.05 compared with siRNA cocktail

Fig. 7

The tube formation inhibition of siRNAs was detected in HUVECs angiogenesis model. a Representative photographs of each treatments were shown (100×). b The total numbers of branching points were decreased treated by siRNA cocktail, VEGF_siR4, and HER_siR3 compared with the untreated and NC_siR (*P < 0.01)