关键词: 环氧化酶2, A549细胞, D-SiRNAs, RNA干扰

Abstract: Objective Here we report the use of Dicer to cleave double-stranded RNA (dsRNA) into small interference RNAs (siRNAs) that can target multiple sites within an mRNA. Methods A549 cells were chosen to be incubated with 5ng/mL IL-1β for different time(0h,3h,6h,9h,12h,) to detect the time-dependent expression of COX-2.To generate the long dsRNA,the COX-2 gene (728bp) was amplified by PCR with a specific forward primer that contained a T7 promoter and a specific reverse primer that contained an SP6 promoter.Then,sense strand RNAs were generated by T7 RNA polymerase and antisense strand RNAs were generated by SP6 RNA polymerase.These single strand RNAs were annealed by the standard method. We mixed dsRNA with Dicer in reaction buffer. The mixture was incubated for 120 min at 37℃.We recovered siRNAs using RNA Purification Column.Transfections with diced siRNAs were performed using the TransMessenger Transfection Reagent in accordance with the manufacturer＇s instructions.COX-2 mRNA and protein were determined by RT-PCR and Western blot respectively. PGE2 was measured by ELISA. Results This study showed that IL-1β induced COX-2 protein expression in A549 cells.We recovered siRNAS that have been generated in vitro by Dicer. D-siRNAs significantly suppress the expression of COX-2 in human pulmonary epithelial .Conclusion In this study,we demonstrate that D-siRNAs significantly suppress the expression of COX-2 in human pulmonary epithelial

Key words: Cyclooxygense-2, A549 cell, D-siRNAs, RNAi