| Objective: Primary liver cancer is one of the most common malignanttumors in our country, surgical resection is the preferred choice and the mosteffective methods. But, the clinical symptoms of liver cancer appear late,many patients with liver cancer are middle or advanced stage when symptomsshow up, rate of surgery is low, prognosis is poor. Metastasis and recurrence isthe most important reason to reduce survival time of patients with liver cancer.Growth and metastasis of tumor depend on angiogenesis. Vascularendothelial growth factor family members(VEGFs) can encourageangiogenesis directly or indirectly. So far, there are seven members in VEGFs,include: VEGF-A, VEGF-B, VEGF-C, VEGF-D, viral VEGF(VEGF-E),snake venom VEGF (VEGF-F) and PLGF(placenta growth factor). ExceptVEGF-E and VEGF-F, other VEGFs members are all encoded by mammalsgene. VEGFs are widely expressed in vertebrate animals, and the codingsequence is highly conservative in different species. VEGF members and its'receptors are over expressed in many tumor tissue, especially in the tumortissue with more blood vessels(such as liver cancer), there is a closerelationship between VEGFs members and characteristics of tumor such asmetastasis and recurrence. As one of VEGF members, VEGF-B expresshighly in various tumor tissue and play an important role in tumor's invasionand transfer. Our previous study suggested that VEGF-B played an importantrole in liver cancer's invasion and metastasis, and have related closely with theprognosis of patients with liver cancer. Therefore, It is expected to inhibit livercancer cell's proliferation and metastasis by restraining VEGF-B express.RNA interference (RNA interference, RNAi) is a new technology developed inrecent years, silenced specific gene in vivo or in vitro. Small interferingRNA (of small interfering RNA, siRNA) is the main effectors of RNA interference. Certain gene can be silenced effectively by targeted siRNA. Inthis study, we reconstruct plasmid carrier of targeted VEGF-B siRNA in vitro,then transfect the vector containing the object gene into liver cancer cell lineHepG-2cell, use reverse transcription-polymerase chain reaction (RT-PCR) todetect VEGF-B mRNA expression between the experimental group and thecontrol group to research the inhibition of targeted VEGF-B siRNA toVEGF-B mRNA exprssion of HepG-2cell. Provide new sites and method forgene therapy of liver cancer and theory basis for further clinical application.Methods: Scaned human VEGF-B coding sequence, designed andcomposed the targeted VEGF-B siRNA, amplified and extracted the carryingplasmid by using E.coli. Human liver cancer cell lines HepG-2cell wascultured in37℃,5%CO2sterile incubator in DMEM culture media containing10%FCS,100U/m1streptomycin and100U/m1penicilin, changed culturemedia depend on cell growth state and color of culture media. Digested cellswith2.5g/L of trypsin when the cells grew logarithmically, then inoculatedthem to six holes training board. We transfected the HepG-2cells by plasmidfollowing the instruction of Lipofectamine2000when the cells covered thetraining board to80%rate, and observed efficiency of transfection withfluorescence microscope after24～48hours. Collected cells and extractedtotal RNA using Trizol Reagents. RT-PCR was used to detected VEGF-B andβ-action(internal stand) espression of experimental group and the controlgroup.Results:1. The targeted VEGF-B siRNA plasmid was amplified by e.coli andextracted successfully.2. Target VEGF-B siRNA transfected the human liver cancer cell lineHepG-2successfully using the plasmid as a carrier and the efficiency oftransfection can reach80%.3. The brightness of each experimental group's electrophoretic bandsshowed different degrees of reduction compared with the control group aftertransfected. The ratio of brightness between VEGF-B and the internal control β-action as follows:0.396±0.037,0.135±0.019,0.271±0.011,0.222±0.030.VEGF-B mRNA expression of each experimental group is lower than thecontrol group Significantly(P<0.05). The expression of VEGF-B mRNA wasinhibited by targeted VEGF-B siRNA in human liver cancer cell linesHepG-2.Conclusions:Targeted VEGF-B siRNA can inhibit expression of VEGF-B mRNA inhuman liver cancer cell line HepG-2cell. |
