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-23’-UTR全长2217bp，上下游引物5’端接xbaI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-23’-UTR不同区域长度的报告质粒（包括3’UTR的全长、前端156bp区域、前端347bp区域、前端1006bp区域、156～347bp区域、157—2217bp区域）。用瞬时转染的方法将含COX-23’-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HT2924h，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156bp区域、前端347bp区域、156～347bp区域分别可将荧光素酶活性下凋70．4％、37．4％、64．8％、24．2％（t=6．13，7．73，9．75，3．92，P〈0．05）。前端1006bp区域、157～2217bp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-23’-UTR156～347bp区域有-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-23’-UTR中含多个调节元件，通过协调作用控制基因表达。
Objective 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 gent expression vectors and pRL-SV40 were co-transfected in HT29 colon cancer ceils 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 lucifcrasc activity were observed in HT29 colon cancer ceils 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.
出处
《中华消化外科杂志》
CAS
CSCD
2009年第6期438-440,共3页
Chinese Journal of Digestive Surgery
基金
无锡市科技发展指导性计划资助（CSZ00736）
