SiRNA Targeted VEGF-B Gene Affects Growth Of Subcutaneous Xenograft Of Liver Cancer Cell In Nude Mouse

objective: Primary liver cancer (PLC) is one of common digestivesystem malignant tumor in our country. The incidence of PLC is insidious, themalignant degree is high and the development is rapid. Most of patients havebeen in the middle or advanced stage when symptoms show up and missoperation opportunity. Although operation is the best and first choice for PLC,it recurrence within2years after operation. Postoperative tumor metastasisand recurrence are the major factors affecting survival time for patients, andtumor angiogenesis is a major cause of tumor growth and metastasis. Vascularendothelial growth factor family (VEGFs) play a key role in the process ofangiogenesis. As one of VEGF members, VEGF-B expresses highly in livertumor and plays an important role in tumor invasion and prognosis of patient.Therefore, it is expected to become a new means for treatment of liver cancerthat inhibits VEGF-B gene expression. RNA interference (RNAi) is a newtechnology of specific gene silence in vivo or in vitro. Small interfering RNA(siRNA) is the main effector of RNA interference, which is small fragmentsconsisted of more than20nucleotides dsRNA molecules and can silenteffectively the specific gene. This study uses RNAi technology, designs andsynthesizes the siRNA targeted VEGF-B gene, transfects siRNA vectors andconstructs subcutaneous xenograft model in nude mouse to observe thegrowth of tumor in experimental group and control group, and to detectVEGF-B protein expression using immunohistochemistry between the twogroups.Methods: We used RNAi technology, synthesized siRNA vectorstargeted VEGF-B in vitro and transfected into human liver cancer cell lineHepG2cells, established the negative group (control group) Eg0and3positive groups (experimental groups) Eg1, Eg2, Eg3, respectively. The cells were stability screened, cultured and amplified using blasticidin, and dividedinto4groups of HepG2cells: three groups of positive plasmid transfectedwith siRNA targeted VEGF-B (Eg1, Eg2, Eg3), one group of negativecontrol plasmid (Eg0group). The treated cells were implanted subcutaneouslyin nude mice to construct subcutaneous xenograft models (named: Ec0, Ec1,Ec2, Ec3, respectively). We observed the growth of xenografts and after30days, the nude mice were sacrificed and the volume and weight of xenograftwere measured. The expression of VEGF-B was detected byimmunohistochemistry staining. Used Image-Pro Plus6.0Image analysissystem for the relative quantitative analysis and calculated integral opticaldensity (IOD) value in unit area of each view positive staining, taked theaverage for statistical analysis.Results:1siRNA vectors targeted VEGF-B were successfully transfected intohuman HepG2cells using plasmid containing fluorescent tag as a carrier andthe efficiency of transfection could reach60~70%.2Xenograft shaped both in control and experimental groups. Thegrowth speed of xenografts in3experimental groups was slower than in thecontrol group. The volumes of xenograft in3experimental groups and controlgroup were (1.107±0.355)cm3，(1.229±0.099)cm3，(1.162±0.188)cm3and(1.698±0.137)cm3, respectively, and he weights of xenograft were(0.696±0.224)g，(0.772±0.062)g，(0.730±0.118)g and (1.068±0.086)g,respectively after30days. The volumes and weights of experimentalxenograft were obviously smaller than in the control group (P <0.05). Nodistant metastasis was observed after carefully dissected.3Immunohistochemical staining showed that3experimental groups (Ec1Ec2Ec3) and control group Ec0were (0.072±0.016)，(0.078±0.004)，(0.075±0.008) and (0.094±0.003), respectively. The expressions of VEGF-Bin3experimental groups were significant lower than in the control group (P<0.05). Conclusion:siRNA vectors targeted VEGF-B gene were successfully transfected intohuman HepG2cells and could obviously inhibit the growth of xenograft andthe expression of VEGF-B protein.