Preliminary Study On Inhibition Of IGF-Ⅱ Expression In Hepatocellular Carcinoma Cells By SiRNAs

Objective: To design and construct siRNAs targeting human IGF-II and observe their influences on the proliferation of cells and inhibition of the expression of IGF-II mRNA in SMMC-7721 cells. To specify the siRNAs sequence and its concentration of the effective gene therapy through siRNAs targeting IGF-II in hapatocellular carcinoma. This paper tries to provide scientific evidence for gene therapy on hapatocellular carcinoma.Methods: Three siRNAs targeting human IGF-II marked with FAM, and one siRNA(siRNA4) without any targeted gene (used for a negetive control) were designed and chemically synthesized. Then the synthetic siRNAs were transfected into SMMC-7721 cells by lipofectamin. The comparison would be made between the siNRAl~3 group and the siRNA4 group, the untransfected group and the lipofectamin group. The efficiency of transfection was examined under fluorescent microscope; the proliferation of cells was evaluated by MTT assay; and the expression level of IGF-II mRNA was detected by RT-PCR.Results: We observed through the FAM that the transfected efficiency was about 65-80%. As compared with the siRNA4 group , the untransfected group and lipofectamin group, the difference of siRNA1-3 group is obvious and noticeable in its MTT value and the surviving rate of cells in each time-points (P<0.01). As the time lengthened, its inhibiting efficiency strengthened, especially for siRNA3. When siRNA3 was used for further concentration experiment, observation continued as the transfected concentration increased to: 10, 20, 40 and 80nM siRNA3 into SMMC-7721 cells respectively by lipofectamin. The result of RT-PCR showed when the concentration reached 10nM, the mRNA began to be dissolved. The inhibiting effect of siRNA3 ascended with the increasing of concentration. The inhibition effect was the best when the concentration was 40nM, and the relative inhibition rate was 86.34%. When 40nM siRNA1-3 were...