摘要目的 探讨环氧合酶2(COX-2)3'-UTR各调控元件在结肠癌细胞株HT29中的调控作用.方法 以HT29细胞的总RNA为模板,采用RT-PCR法扩增COX-2 3'-UTR全长2217 bp,上下游引物5'端接xba Ⅰ酶切位点.通过PCR制备3'端缺失体方法结合荧光素酶报告基因技术构建含COX-2 3'-UTR不同区域长度的报告质粒(包括3'UTR的全长、前端156 bp区域、前端347 bp区域、前端1006 bp区域、156～347 bp区域、157～2217 bp区域).用瞬时转染的方法将含COX-2 3'-UTR不同区域长度的报告质粒和内参pRL-SV40质粒共转染结肠癌细胞株HT29 24 h,然后分别测定荧光素酶相对活性.数据分析采用t检验.结果 3'-UTR的全长、前端156 bp区域、前端347 bp区域、156～347 bp区域分别可将荧光素酶活性下调70.4%、37.4%、64.8%、24.2%(t=6.13,7.73,9.75,3.92,P<0.05).前端1006 bp区域、157～2217 bp区域对荧光素酶活性无明显影响(t=0.13,0.01,P>0.05).结论 在COX-2 3'-UTR 156～347 bp区域有一重要调控元件可与ARE元件共同作用下调目的基因表达.结肠癌细胞株HT29中COX-2 3'-UTR中含多个调节元件,通过协调作用控制基因表达.

abstractsObjective To investigate the regulative effect of cyclooxygenase-2 (COX-2) 3'-UTR in HT29 colon cancer cells. Methods Total RNA was extracted from HT29 colon cancer cells and used as a template to amplify COX-2 gene by reverse transcription polymerase chain reaction. Using PCR technique to construct a series of luciferase reporter gene expression vectors containing various regions of the 3'-UTR of COX-2 (including the whole gene region, gene region between 1 to 156 bp, gene region between 1 to 347 bp, gene region between 1 to 1006 bp, gene region between 156 to 347 bp and gene region between 157 to 2217 bp). These reporter gene expression vectors and pRL-SV40 were co-transfected in HT29 colon cancer cells by Lipofectamine 2000. The relative luciferase activity of the HT29 colon cancer cells was tested. All data were analyzed via t test. Results The luciferase activity was reduced by 70.4%, 37.4%, 64.8% and 24.2% in HT29 colon cancer cells transfected with the whole COX-2 gene region, gene region between 1 to 156 bp, gene region between 1 to 347 bp and gene region between 156 to 347 bp, respectively (t = 6.13, 7.73, 9.75, 3.92, P < 0.05). No obvious changes of luciferase activity were observed in HT29 colon cancer cells transfected with gene regions between 1 to 1006 bp and between 157 to 2217 bp (t = 0.13, 0.01, P > 0.05). Conclusions A region between 156-347 bp in the 3'-UTR of COX-2 has been found which can down-regulate the expression of COX-2 with the cooperation of the ARE element. The 3'-UTR of COX-2 contains several control elements that regulate the expression of COX-2 in colon cancer cells.