D-siRNAs抑制A549细胞COX-2表达

D-siRNAs suppresse expression of COX-2 in A549 cells

目的长片段双链COX-2RNA(dsRNAs)在体外经Dicer酶消化获得小RNA(D-siRNAs)抑制A549细胞COX-2的表达。方法(1)IL-1β5 g/L干预A549细胞,Western blot法观察其COX-2蛋白表达的时间依赖性。(2)Trizol法抽提A549细胞总RNA,RT-PCR扩增COX-2基因。(3)设计两端带T7 promoter,SP6 promoter的COX-2cDNA的PCR引物,通过PCR方法,获得两端带T7及SP6 promoter的COX-2DNA。(4)用RiboMAX Large Scale RNA Produc-tion Systems-SP6 and T7试剂盒体外将COX-2DNA转录为RNA。(5)长片段COX-2RNA经Dicer酶消化为小RNA(D-siRNAs)。(6)用TransMessenger Transfection Reagent将D-siRNAs转染A549细胞。(7)COX-2mRNA表达用RT-PCR检测,细胞培养上清液中PGE2含量用ELISA测定。结果(1)IL-1β干预A549细胞9 h后COX-2表达到高峰。(2)在体外,长片段COX-2dsRNAs经Dicer酶消化获得D-siRNAs。(3)D-siRNAs可有效抑制A549细胞COX-2的表达并降低PGE2的分泌。结论长片段双链COX-2RNA(dsRNAs)在体外经Dicer酶消化获得小RNA可有效抑制A549细胞COX-2的表达。

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-1β 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 anti- sense 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 ac- cordance 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-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 D-siRNAs significantly suppress the expression of COX-2 in human pulmonary epithelial.