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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 type homeobox transcription factor 2 (CDX2) promotes proliferation of colorectal cancer cells and has tumorigenic potential. However, little has been done on the regulatory mechanisms of CDX2 translation. In this study, based on the results of transfection and enzyme activity analysis, it was first demonstrated that internal ribosome entry site (IRES) exists in 5’untranslated region (5’-UTR) of CDX2, Mediates translation initiation in HEK293 cells as well as in tumor cell lines such as liver cancer, colon cancer and ovarian cancer. Unlike the classic cap-dependent translation, the IRES-mediated translation mechanism does not require a cap-like structure under the influence of some IRES trans-acting factors (ITAFs) and the IRES can be directly Recruiting small subunits of ribosomes, initiating translation. At the same time, IRES-mediated translation in tumor cells may be one of the reasons leading to abnormal protein expression. The expression of CDX2 in four tumor cell lines was detected by Western blotting. It was found that the IRES activity of CDX2 was positively correlated with its protein expression in tumor cells, and its activity was the highest in the resistant ovarian tumor cell lines. In addition, analysis of the 5 ’end of the CDX2 IRES element and analysis of reporter enzyme activity demonstrated that the active center of the CDX2 5’-UTR is between 68 and 147 bases and 67 bases at the 5’ end form a distal stem Loop, inhibits full-length IRES activity. Experimental results of site-directed mutagenesis of 68-147 active regions revealed that three stem-loop domains (68GCCGGC73, 88GCCAG92 and 114CUCUG118) are integral to IRES elements. The above results demonstrate that the CDX2 5’-UTR has IRES activity and its activity depends on the secondary stem-loop structure. Therefore, specific stem-loop domains can be studied to make them the target of oncology drugs.