D-siRNAs suppresse expression of COX-2 in A549 cells / 基础医学与临床

ABSTRACT

Objective To cleave double-stranded RNA(dsRNA) into small interference RNAs(siRNAs) that can target multiple sites within an mRNA.Methods A549 cells were isolated to incubate with 5 g/L IL-1? for different times to detect the time-dependent expression of COX-2.To generate the long dsRNA,the COX-2 gene(728 bp) 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 strands RNAs were generated by SP6 RNA polymerase.These single strands RNAs were annealed by the standard method.We mixed dsRNA with Dicer in reaction buffer.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 IL-1? 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 D-siRNAs significantly suppress the expression of COX-2 in human pulmonary epithelial.