| Objective To construct recombinant plasmids generating small interfering RNA in mammalian cells,and investigate its effect on the expression of Trop2 mRNA and protein in human gastric cancer BGC823 cells,as well as the effects of recombinant plasmids on migration and invasion of gastric cancer BGC823 cells.Methods Three kinds of constructed recombinant plasmids generating small interfering RNA, including three interferential sequences pGenesill.1-Trop21,pGenesill.1-Trop22, pGenesill.1-Trop23, were designed and used to transfect BGC823 cells by LipofectamineTM 2000. The transfection efficiency was observed under fluorescence confocal microscopy.Trop2 mRNA and Trop2 protein were analyzed by real-time fluorescence quantitative PCR and Western blot,respectively. Transwell assay was performed to assess the effect of Trop2 targeted RNA interference on the migratory and invasive properties of gastric cancer BGC823 cells.Results (1)Enzyme digestion analyzing and DNA sequencing showed that Trop2 targeted RNA interference recombinant plasmids were constructed successfully. (2)The transfection conditions and the rate of transfection:the most highly efficiency was 75% when the ratio of recombinant plasmids to LipofectamineTM2000 is 1:4.(3)Real-time fluorescence quantitative PCR and Western blot results:All three recombinant plasmids downregulated the expression of Trop2 mRNA and protein. The inhibition ratios on Trop2 mRNA expression of three plasmids reached to 23.9%,23.7%,29.9%, respectively, which were significantly different to the control group (P<0.05). The inhibition ratios on Trop2 protein expression of three plasmids reached to 32.8%,25.5%,62.0%, respectively. The difference to the control group was significant(P<0.05).The most effective recombinant plasmid pGenesil1.1-Trop23 was selected for the following experiments.(4)Transwell migration and invasion assay results:48 hours after transfection of recombinant plasmid pGenesill.1-Trop23,the numbers cells of through matrigel membrane were 43±6 and 23±5,with significant difference to the control group(P<0.05).Conclusion The constructed recombinant plasmids can suppress the expression of Trop2 mRNA and protein in gastric cancer BGC823 cells. Transfection with the recombinant plasmid pGenesill.1-Trop23 may inhibit the migration and invasion of BGC823 cells. It has laid a solid foundation for further study of antineoplaston targeted to Trop2 in gastric cancer. |
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