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内部核糖体进入位点(IRES)调控多种癌细胞系中尾型同源盒转录因子2的翻译

Internal Ribosomal Entry Site Regulates CDX2 Translation in Multiple Cancer Cell Lines
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摘要 尾型同源盒转录因子2(caudal type homeobox transcription factor 2,CDX2)促进肠癌细胞增殖,并具有致瘤的潜能。然而,对于CDX2翻译水平的调控机制研究甚少。本研究基于转染及报告酶活性分析结果,首次证明CDX2 5'非翻译区(5'untranslated region,5'-UTR)存在内部核糖体进入位点(internal ribosome entry site,IRES),并通过IRES在HEK293细胞以及肝癌、肠癌和卵巢癌等肿瘤细胞株中介导翻译起始。IRES介导的翻译机制与经典的帽依赖翻译途径(cap-dependent translation)不同,在一些IRES反式作用因子(IRES trans-acting factors,ITAFs)的作用下,不需要帽状结构,IRES可以直接招募核糖体小亚基,起始翻译。同时,肿瘤细胞内IRES介导的翻译方式可能是引起蛋白质异常表达的原因之一。通过Western印迹检测CDX2在4种肿瘤细胞系中的表达,发现CDX2的IRES活性与其在肿瘤细胞中蛋白质表达量正相关,其活性在耐药型卵巢肿瘤细胞株中最高。此外,CDX2 IRES元件的5'端截短及报告酶活性分析证明,CDX2 5'-UTR的活性中心位于68-147碱基之间,而位于5'末端的67个碱基形成一个远端茎环,抑制全长IRES的活性。定点突变68-147活性区域的实验结果揭示,3个茎环结构域(68GCCGGC73,88GCCAG92和114CUCUG118)是IRES元件中不可或缺的部分。上述结果证明,CDX2 5'-UTR具有IRES活性,其活性依赖于二级茎环结构。因此,可以针对特殊的茎环结构域进行研究,使之成为肿瘤药物作用靶点。 The caudal-related homeobox transcription factor( CDX2) enhances proliferation and has tumorigenic potential in human colon cancer cell lines. However,a very few studies focus on the translation regulation of CDX2 mRNA. In this study,we demonstrated that translation of CDX2 is controlled by an internal ribosome entry site( IRES) located in the 5'-untranslated region( UTR) of CDX2 mRNA. The IRES allows for efficient CDX2 translation in HEK293 and human hepatocellular,colorectal and ovarian cancer cell lines. Different from the classical cap-dependent translation mechanism,IRES could recruit the 40 S ribosomal subunit and initiate translation through its interaction with an additional complex set of IRES trans-acting factors( ITAFs). Meanwhile, IRES-mediated translation mechanism may be one of the causes for protein over-expression in cancer cells. We found thatCDX2 IRES activity was highest in A2780 / PTX that also over-expressed the CDX2 protein,indicating a positive correlation between CDX2 IRES activity and its protein expression. After mapping the entire CDX2 5'-UTR,we determined that the region between 68 and 147 is indispensable for the highest IRES activity. Contrarily,a distal stem-loop between 1 and 67 remarkably inhibited the activity of CDX2 IRES. Moreover,we performed mutational analysis on the functional region of CDX2 5'-UTR and found three stem-loops(68GCCGGC73,88GCCAG92and114CUCUG118) were essential for its IRES activity. In conclusion,we showed that CDX2 5'-UTR exhibited IRES activity,which was dependent on its secondary structure. It is possible that the IRES stem-loop structure may be a potential target for cancer treatment.
作者 高文青 李琪 陈蕴 朱瑞宇 金坚 GAO Wen-Qing, LI Qi, CHEN Yun, ZHU Rui-Yu, JIN Jian (Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, J iangsu , China)
出处 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2017年第3期260-268,共9页 Chinese Journal of Biochemistry and Molecular Biology
基金 国家自然科学基金(No.81101667) 江苏省自然科学基金(No.BK2009071)资助
关键词 尾型同源盒转录因子2 5'非编码区 内部核糖体进入位点 caudal type homeobox transcription factor 2(CDX2) 5'-untranslated region(5'-UTR) internal ribosome entry site(IRES)
作者简介 联系人Tel:510-85918219;E-mail:jinjian31@126.com ry_zhu@sina.com
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