| Objective: To design and select effective siRNA targeting human IGF-II and to construct thevector of recombinant RNA polymerase II hAFP/hTERT dual promoters-driven siRNAexpression against human IGF-II gene.Method:①According to the siRNA design principle, three siRNAs (siRNA1、siRNA2、siRNA3) targeting IGF-II mRNA and one negative control siRNA （NC siRNA）were designedand synthesized.25nM,50nM,100nM three different concentrations of siRNA1-3weretransfected into Huh7cells through Lipofectamine Transfection Reagent. The level of IGF-IImRNA expression in Huh7cells were detected by quantitative reverse transcription-polymerasechain reaction (qRT-PCR) at24h after transfection to select the most effective siRNA and its bestinhibition concentration;②The hAFP and hTERT promoter core sequence were cloned into thepGL3-basic vector to establish the vector of recombinant RNA polymerase II hAFP/hTERT dualpromoters-driven siRNA expression against human IGF-II gene.Results:①The result of qRT-PCR showed the expression levels of IGF-II mRNA in Huh7cellstransfected with siRNA1-3were significantly declined, and the inhibition efficiency was about67.18%-94.82%. The inhibition effect of siRNA3was the best when the concentration was25nM, and the relative inhibition rate was94.82%.②The hAFP and hTERT promoter coresequence fragments were successfully amplified from AC16cells, and were269bp,456bp,respectively. The hAFP and hTERT promoter core sequences connected with siRNA3wererecombined to establish the vector of pGL3-hAFP-hTERT-siRNA3. No deletion and mutationwere examined in vectors as compared with the hAFP and hTERT promoter core sequence wereaccordant with GenBank data.Conclution:①The most effective siRNA3sequences against human IGF-II was selected andthe best inhibition concentration was25nM;②The vector of recombinant RNA polymerase IIhAFP/hTERT dual promoters-driven siRNA expression against human IGF-II gene weresuccessfully constructed. |
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