Influences Of VEGF-B Gene Targeting SiRNA On The Expression Of VEGF-B Protein In Nude Mouse Subcutaneous Xenograft

Objective: Primary liver cancer (PLC) is one of common malignanttumors. The incidence of PLC is insidious and the progress is fast. Thetreatments of PLC include surgical resection, radiofrequency ablation,microwave curing, chemoembolization (TACE), radiation therapy, biologicalimmune therapy and so on. The mainly treatment for PLC is surgical resection,however, the postoperative5-year survival rate of patients is low because ofthe postoperative tumor recurrence and metastasis. Tumor growth depends onangiogenesis, vascular endothelial growth factor (VEGF) is an importantregulator of angiogenesis which can promote angiogenesis and lymphangio-genesis, regulate lipid metabolism, protect nerve, and is closely related totumorigenesis and development. There are7members in VEGF family, inwhich VEGF-B is an important member. Our preliminary studies revealedVEGF-B related to PLC incidence and development. RNA interference (RNAi)is post-transcriptional gene silencing which can silences the targeted gene withhigh specificity and high efficiency. siRNA is the main mediator of RNAi. Inthis study, we selected3targets in human VEGF-B gene, constructed3VEGF-B gene targeting vectors in vitro. The siRNA vectors were transfectedinto HepG2cells and constructed subcutaneous xenograft models in nudemice. We study the effect of VEGF-B gene targeting siRNA on the expressionof VEGF-B in nude mouse subcutaneous xenograft and provides a new targetand theoretical basis for PLC.Methods: We selected3targets in human VEGF-B gene, constructed3siRNA vectors targeting to VEGF-B in vitro. Human HepG2cells wereconventional cultured, three siRNA and control vectors were transfected intoHepG2cells (named Bt0, Bt1, Bt2, Bt3respectively after transfection).Transfection efficiency was observed after48h. Cells were stability screened, cultured and amplified using blasticidin. Twenty four nude mice wererandomly divided into4groups. The treated cells were implantedsubcutaneously in nude mice to construct subcutaneous xenograft models(named: mBt0, mBt1, mBt2and mBt3respectively). Nude mice wereconventionally bred and the growth of xenograft was observed. After30days,the nude mice were sacrificed and the volume and weight of xenograft weremeasured. The expression of VEGF-B was detected by Western blot.Results:1siRNA vectors targeting to VEGF-B were successfully transfected intoHepG2cells with a70~75%rate after48h.2Xenograft shaped both in control and experimental groups. The growthrate of xenografts in3experimental groups was slower than in the controlgroup. The volumes of xenograft were (1.671±0.131)cm3,(1.083±0.224)cm3,(1.252±0.204)cm3and (1.127±0.276)cm3respectively after30days. Theweights of xenograft were (1.049±0.104)g,(0.681±0.130)g,(0.804±0.119)gand (0.733±0.138)g respectively. The volumes and weights of experimentalxenograft were obviously smaller than in the control group (P<0.05). Nodistant metastasis was observed after carefully dissected.3Western blot analysis showed that the stain stripes in3experimentalgroups were darker than in the control group. The gray values of VEGF-Bwere0.692±0.059,0.411±0.039,0.544±0.063and0.503±0.102, respectively.The expressions of VEGF-B in3experimental groups were lower than in thecontrol group (P<0.05).Conclusions: siRNA vectors targeting to VEGF-B were successfullytransfected into HepG2cells and subcutaneous xenograft models wereconstructed. The growth of xenograft and the expression of VEGF-B wereobviously inhibited.