The Study Of SiRNA Targeting VEGF-B To Treat Primary Liver Cancer

Objective: Primary liver cancer (PLC) is one of the most commonmalignant tumors in clinical, which is frequently associated with metastasis inearly stage and has a high rate of recurrence after operation or interventiontreatment. PLC is the5th commonest malignancy worldwide and is the thirdmost common cause of cancer-related death. In our country, incidence of PLCaccount for55%of the world. In present, operation is the best and first choicefor PLC although with a poor effect and a high rate of recurrence within twoyear after operation, and is only suit for parts of early stage patients about10%³0%. To the patients with advanced stage PLC, chemoembolization alsomake a poor prognosis. In recent years, biotherapy, especially gene targetedtherapy showed a specific advance in inhibiting proliferation, preventing anddelaying recurrence of PLC, and improved the life quality of patients. It hasbecome a focus of research.Vascular endothelial growth factor (VEGF) is a growth factor family.Their members have a similar structure. In the present, the family contains7members: VEGF-A (also known as VEGF), VEGF-B, VEGF-C, VEGF-D,VEGF-E, VEGF-F and PLGF (Placenta growth factor). All of them areglycoprotein with a similar dimeric crystal structure. VEGFs have the effectsthat promote proliferation of endotheliocyte, increase permeability ofbasement membrane and induce the process of neoangiogenesis by binding totheir specific receptors in blood and lymphatic vessels. VEGFs paly a veryimportant role in growth and metastasis process of malignant tumor. A numberof reseach have showed that almost all of the malignant tumor has theexpression of VEGFs. In contrast, there is no or only a very low expressionlevel of VEGFs in normal tissues.RNA interference is a phenomenon that sequencing-specific post-transcr- iptional gene silencing mediated by double-stranded RNA in the creature. It isa process that targeted mRNA blocked by the double-stranded RNA. The longstranded RNA (dsRNA) synthesized in vitro has a non-specific cytotoxiceffect. However, Small/short interfering RNA (siRNA,21²3nt) can avoidthis non-specific cytotoxic effect. Some reseach showed that the siRNAsynthesized in vitro have the same function that breakdown targeted mRNAjust as the endogenous siRNA. As we know more about the mechanism oftumor and the development of biotechnology, gene targeted therapy becomemore and more important in tumor therapy. RNA interference technology hasshowed its potential value for clinical application in the treatment of tumorand has been paid close attention in aspect of gene function and targetedtherapy.Method: HepG2(Human hepatocellular liver carcinoma cell lines) cellwas cultured in conventional way. To choose three target sites based onVEGF-B gene sequence of human (NM₀03377.3), design and synthesizethree siRNA plasmid vectors (S1, S2, S3) targeting the three chosen VEGF-Bgene sites, recpectively, and synthesize a negative control plasmid vector(NC). These four kinds of plasmid vector can express green fluorescenceprotein (GFP). The siRNA plasmid vectors (S1, S2, S3) and empty plasmidvector (NC) were transfected into HepG2cells using lipofectamine2000reagent, recepectively. We evaculated the transfection rate by laser scanningconfocal microscope. After transfection for48hours, the expression ofVEGF-B protein were detected with western blot, respectively.Results: Western blot analysis showed that electrophoretic bands ofVEGF-B had a reduction in groups of HepG2-S1, HepG2-S2and HepG2-S3compared with HepG2-NC group. VEGF-B protein level in HepG2-NC,HepG2-S1, HepG2-S2and HepG2-S3group were0.547±0.012,0.322±0.030,0.235±0.008and0.272±0.018, respectively. Expression of VEGF-Bprotein level in three experiment groups showed a marked reduction comparedwith the HepG2-NC control (P<0.05). Population mean between eachexperiments compared to the control showed a statistical significance (P<0.05). Therefore, HepG2-S1, HepG2-S2and HepG2-S3interfering vectorsall could inhibit expression of VEGF-B protein in HepG2cell. HepG2-S2hadthe strongest inhibition rate, the second was HepG2-S3, and HepG2-S1wasrelatively weaker.Conclusion: The siRNA targeting VEGF-B designed in vitro can inhibitexpression of VEGF-B protein of liver cancer cell line hepG2cell.