ABSTRACT
Objective To investigate of human esophageal squamous carcinoma cells(Eca-109) in which cyclooxygenase (COX)-2 gene was knocked down by adenovirus delivered siRNA.Methods Based on the plasmid pSUPER cloned with RNA polymerase Ⅲ-dependent promoter HI,the interfering plasmid psiRNA/COX-2 targeting human COX-2 mRNA was constructed,The siRNA/COX-2 fragment was derived from psiRNA/COX-2 digested by Not I and Xho 1.and was cloned into the shuttle plasmid pAdTrack.Then pAdTrack/siRNA/COX-2 was obtained and co transfected into the E.coli strain BJ5183 with the bone plasmid pAdEasy-1,the recombinant adenovirus Ad/siRNA/COX-2 was generated by homologous recombination.Having been packaged and amplified in cells 293,Ad/siRNA/COX-2 was transfected into Eca-109 cells.The PGE2 concentration in the cells culture supernatant was determined by ELISA,and the level of COX-2 mRNA in the cells was tested by real time PCR.Moreover,cell cycle and apoptosis were determined by flow cytometry,and cells growth curve was protracted.Results The recombinant adenovirus Ad/siRNA/COX-2 was successfully constructed.Ninty six hours after Ad/ siRNA/COX 2 transfecting into Eca 109 cells,COX-2 mRNA was reduced by 71.7%,and PGE2 concentration in the cells culture supernatant was decreased by 62.0%.Correspondingly,the growth of cells slowed down.At the same time,the cells in G0-G1 phase was increased by 32.24%,and those in S phase and G2-M phase were reduced by 16.38% and 15.86%.respectively.And cells apoptosis index was increased by 9.19%.Conclusion The adenovirus based-RNAi was capable of knocking down remarkably COX-2 of human esopbageal carcinoma cells,which lead to growth of cells slowing down.