Construction And Evaluation Of MicroRNA-141and ShRNA Recombinant Plasmids Targeting Mouse HMGB1Gene

Objective:To construct effective microRNA-141and shRNA (short hairpin) recombinant plasmids targeting mouse HMGB1gene.Methods:1. the microRNA-141sequence was artificially synthesized and successfully inserted into the pYr-miR-30-shRNA plasmid. Restriction endonuclease analysis and DNA sequencing confirmed the sequence of the recombinant plasmid pYr-adv-mmu-miR141. then the recombinant plasmid pYr-adv-mmu-miR141was transfected into mouse hepatoma cell line Hepa1-6for shRNA screening. HMGB1knockdown efficiency was evaluated by Q-PCR and Western blot analysis.2. Four pairs of shRNA sequences targeting mouse HMGB1gene and one pair of negative control shRNA sequence were designed and synthesized. The recombinant plasmids were constructed and identified by Xhol restriction enzyme. Then the four plasmids were transfected into mouse hepatoma cell line Hepal-6for shRNA screening. HMGB1knockdown efficiency was evaluated by Q-PCR and Western blot analysis.Results:1. Restriction enzyme digestion and DNA sequencing demonstrated that the recombinant plasmid pYr-adv-mmu-miR141was successfully constructed. Moreover, the expression of HMGB1in mouse hepatoma cell line Hepa1-6infected with the the recombinant plasmid pYr-adv-mmu-miR141was significantly decreased (p<0.05).2. Restriction enzyme digestion and DNA sequencing demonstrated that the recombinant plasmid pYr-adv-mmu-miR141was successfully constructed. However, the expression of HMGB1in mouse hepatoma cell line Hepa1-6infected with the the recombinant plasmid pYr-1.1-musHMGB1sh1～4were not significantly decreased (p>0.05).Conclusions:1.The recombinant plasmid pYr-adv-mmu-miR141targeting mouse HMGB1gene was successfully constructed. The expression of HMGB1was significantly suppressed by the plasmid pYr-adv-mmu-miR141.2. The sequences of plasmid HMGB1sh1～4were successfully constructed.However, the expression of HMGB1was not significantly suppressed by the plasmid pYr-1.1-musHMGB1sh1～4.