Jiang Shun-Yuan, Wu Meng-Shiun, Chen Liang-Ming, Hung Mei-Whey, Lin Huai-En, Chang Gu-Gang, Chang Tsu-Chung
Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, ROC.
Biochem Biophys Res Commun. 2005 Jun 3;331(2):630-9. doi: 10.1016/j.bbrc.2005.03.214.
The expression of retinoic acid-induced gene 1 (RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all-trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5'-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the -4910/-5509 fragment of the 5'-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between -5048 and -5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5'-TGACCTctattTGCCCT-3' (DR5, -5243/-5259), and an inverted repeat sequence with six nucleotide spacing, 5'-AGGCCAtggtaaTGGCCT-3' (IR6, -5323/-5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the atRA-mediated induction. Taken together, we have identified and characterized the functional atRA response element that is responsible for the atRA-mediated induction of RIG1 gene.
维甲酸诱导基因1（RIG1）是一种II类肿瘤抑制基因，其表达在经类视黄醇处理的细胞中被诱导。RIG1已被证明在全身广泛表达，该基因表达的增加似乎会抑制细胞增殖。最近的研究还表明，该基因可能在细胞分化和癌症进展中发挥重要作用。尽管这种蛋白质具有显著的调节作用，但类视黄醇诱导RIG1表达的分子机制仍有待阐明。本研究旨在探讨全反式维甲酸（atRA）介导的RIG1基因表达诱导的分子机制。采用聚合酶链反应生成了总共10个荧光素酶构建体，这些构建体包含RIG1 5'-基因组区域的各种片段。然后将这些构建体转染到人胃癌SC-M1细胞和乳腺癌T47D细胞中进行反式激活分析。相对于翻译起始位点，atRA仅通过RIG1基因5'-基因组区域的-4910/-5509片段显著诱导荧光素酶活性。对该启动子片段的进一步分析表明，主要的atRA反应区域位于RIG1基因的-5048至-5403之间。在该区域内，鉴定出一个具有五个核苷酸间隔的直接重复序列，5'-TGACCTctattTGCCCT-3'（DR5，-5243/-5259），以及一个具有六个核苷酸间隔的反向重复序列，5'-AGGCCAtggtaaTGGCCT-3'（IR6，-5323/-5340）。DR5元件的缺失和突变消除了atRA介导的活性，但IR6元件没有。用atRA处理的细胞的核提取物进行的电泳迁移率变动分析表明，维甲酸受体（RAR）和类视黄醇X受体（RXR）异二聚体特异性结合到该反应元件上。除了功能性DR5外，该区域还包含许多其他潜在的序列元件，这些元件是使atRA介导的诱导最大化所必需的。综上所述，我们已经鉴定并表征了负责atRA介导的RIG1基因诱导的功能性atRA反应元件。
