Construction And Primary Selection Of The MicroRNA Recombinant Plasmids In The Gene Therapy Of Nasopharyngeal Carcinoma

Objective: Purpose of this study is intended to design artificial miRNAs by simulating native miRNA and to construct the miRNA recombinant plasmids, which targeted the hTERT and VEGF gene respectively. The inhibitory abilities on gene expressions of these designed miRNAs were verified in nasopharyngeal carcinoma tentatively, and the most effective plasmids were selected for the further invo and vitro experiments.Methods: Computer-designed oligo nucleotide sequences which express the pre-miRNA were connected to plasmid pcDNA6.2-GW/Em-GFP-miR after annealing, then the recombinant plasmids would be sent to company to fulfill the sequences analysis. Fluoromicroscopy was applied to observe the transfectional effect after the plasmids enter CNE-2 cells via lipofectamine. The quantity of target gene expression could be detected by RT-PCR and Western blotting.Results: Three recombinant plasmids i.e. pcDNA6.2-GW/Em-GFP-miR-hTERT2035, pcDNA6.2-GW/EmGFP-miR-hTERT3010 and pcDNA6.2-GW/EmGFP-miR-hTERT3100 which targeted hTERT and other three recombinant plasmids i.e. pcDNA6.2-GW/EmGFP-miR-VEGF1022, pcDNA-6.2-GW/EmGFP-miR-VEGF1025, pcDNA6.2-GW/ EmGFP-miR-VEGF1035, which aimed VEGF were constructed. The consequence of sequence analysis of those plasmids was coincided with computer designs. Green fluorescence could be observed in post-transfectional CNE-2 cells, which convinced that cell transfections were successful, and transfected plasmids were expressed in cells. By RT-PCR, it was observed which expression of mRNA of cells transfected by pcDNA6.2-GW/EmGFP-miR-hTERT2035 and pcDNA-6.2-GW/EmGFP-miR-VEGF1025 was obviously down-regulated. Western blotting showed that the protein bands of target genes in all kinds of transfected cells were decreased and it implied that protein synthesis was inhibited. Results found that pcDNA6.2-GW/EmGFP-miR-hTERT2035 and pcDNA-6.2-GW/EmGFP- miR-VEGF1025 plasmids were more effective. Therefore, the site for hTERT2035~2055 and site for VEGF1025-1045 were the most optimal sites for inhibiting respectively hTERT and VEGF gene expression.Conclusions: Six kinds of recombinant plasmids were constructed successfully and two most effective plasmids were selected, which provided the elementary sources for the further study of miRNA- based gene therapy in nasopharyngeal carcinoma.