重组腺病毒Ad/siRNA/环氧合酶-2的构建及其抑制食管癌细胞的效果

Effects of cyclooxygenase-2 silencing by adenovirus-delivered siRNA on the relevant changes of human esophageal carcinoma cells

目的观察腺病毒介导的 RNA 干扰(RNAi)技术对人食管鳞癌细胞(Eca-109)环氧合酶(COX)-2基因表达的抑制效果及其对 Eca-109细胞生长的影响。方法采用含 RNA 聚合酶Ⅲ启动于H1的质粒 pSUPER 构建针对人 COX-2 mRNA 的重组质粒 psiRNA/COX-2。Not Ⅰ和 Xho Ⅰ双酶切psiRNA/COX-2得到 siRNA/COX-2表达片段,定向克隆至腺病毒穿梭质粒 pAdTrack 的相同位点得到pAdTrack/siRNA/COX-2,后者与骨架质粒 pAdEasy-1在大肠杆菌 BJ5183中同源重组得到腺病毒 Ad/siRNA/COX-2,经293细胞包装、扩增后转染 Eca-109细胞。ELISA 法测定培养液上清的前列腺素(PG)E2浓度,PCR 法检测细胞 COX-2 mRNA 水平,流式细胞仪检测细胞凋亡与周期分布,绘制细胞生长曲线。结果重组腺病毒 Ad/siRNA/COX-2构建成功,转染 Eca-109细胞后,细胞 COX-2 mRNA 水平下降71.7%(P<0.01),培养液上清 PGE2浓度下降62.0%(P<0.01);细胞生长明显减缓,GO-G1期细胞比例增加、S 期与 G2-M 期细胞比例减少(P<0.01),细胞凋亡增多(P<0.01)。结论腺病毒介导的 RNAi 技术可显著抑制人食管鳞癌细胞 COX-2基因表达.从而导致肿瘤细胞增殖减缓、凋亡增加。

Objective To investigate of human ese）phageal 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 H1, 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 hy Not Ⅰ and Xho Ⅰ, and was cloned into the shuttle plasmid pAdTrack. Then pAdTrack siRNA C（）X-2 was obtained and co transfeeted 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 protraeted. 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 ins phase and G2-M phase were reduced by 16. 38% and 15. 86%,respectively. And cells apoptosis index was increased hy 9. 19%. Conclusion The adenovirus hased-RNAi was capable of knocking down remarkably COX-2 of human esophageal carcinoma cells, which lead to growth of ceils slowing down.