Translational regulation of aurora-A kinase expression and its implication for the progression of gynecological cancers.

极光 A 激酶表达的翻译调控及其对妇科癌症进展的影响。

• 批准号: 17591766
• 负责人: MORINAGA Tomonori
• 金额: $ 2.24万
• 依托单位: Hyogo College of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591766/
• 关键词: Cell Cycle; M Phase; Translation regulation; Gene Expression; Cancer-related Genes

The over expression of aurora-A kinase is thought to bring unequal distribution of chromosomes to daughter cells causing aneuploidy, and is implicated in progression of cancers. In addition to increase in the amount of mRNA, cancer cells overproducing aurora-A express larger transcripts that are not found in normal cells. The large aurora-A mRNA isoforms have longer 5'-untranslated region (5'-UTR) and are derived from alternative use of the noncoding exons. In this work we analyzed the effects of 5'-UTR on the promoter activity to investigate possible relationship between expression of the isoforms and the overproduction of the kinase in cancer cells.We constructed bicistronic reporter plasmids by inserting PCR-amplified 5'-UTR fragments between firefly luciferase gene derived from pGL3-Basic vector and renilla luciferase gene derived from phRL-CMV vector. The plasmids were transfected into cultured cancer cells to see whether the 5'-UTR fragments carry an internal ribosome entry site … More (IRES). Although the results showed an absence of IERS in the 5'-UTR, we found that 5'-UTR fragments strongly down-regulate expression of the first (upstream) cistron.The extent of down-regulation of the second (downstream) cistron depended on the cells used suggesting complex regulatory mechanisms.To simplify system, we inserted the 5'-UTR fragments to pGL3-Control vector, between SV40 promoter and the luciferase gene, to produce monocistronic reporter vector. We found that the 5'-UTR fragments down-regulated the reporter gene expression confirming the presence of negative regulatory elements in the 5'-UTR. To determine the expression-suppressive domains (SDs), sequential deletion was introduced into the 5'-UTR from both 5'-and 3'-ends. This deletion experiments identified two SDs affecting the reporter gene expression. These SDs were inserted to pGL3-Control vector upstream or downstream of SV40 promoter to see if the suppressive effect is reproducible with the short DNA fragments. We found that the SDs repressed expression when placed downstream of a promoter. Furthermore, it was found that the 5'-UTR carry a promoter activity in its 5'-end region. Less

极光-A 激酶的过度表达被认为会导致子细胞染色体分布不均匀，从而导致非整倍性，并与癌症的进展有关。除了 mRNA 数量增加之外，过量产生 aurora-A 的癌细胞还表达正常细胞中未发现的较大转录物。大的 aurora-A mRNA 亚型具有更长的 5'-非翻译区 (5'-UTR)，源自非编码外显子的替代使用。在这项工作中，我们分析了 5'-UTR 对启动子活性的影响，以研究癌细胞中异构体的表达与激酶过量产生之间可能的关系。我们通过在 pGL3-Basic 载体衍生的萤火虫荧光素酶基因和 phRL-CMV 衍生的海肾荧光素酶基因之间插入 PCR 扩增的 5'-UTR 片段构建双顺反子报告质粒 矢量。将质粒转染到培养的癌细胞中，以观察 5'-UTR 片段是否携带内部核糖体进入位点 (IRES)。虽然结果显示 5'-UTR 中不存在 IERS，但我们发现 5'-UTR 片段强烈下调第一个（上游）顺反子的表达。第二个（下游）顺反子的下调程度取决于所使用的细胞，表明复杂的调节机制。为了简化系统，我们将 5'-UTR 片段插入到 pGL3-Control 载体中，位于 SV40 启动子和 荧光素酶基因，产生单顺反子报告载体。我们发现 5'-UTR 片段下调报告基因表达，证实 5'-UTR 中存在负调控元件。为了确定表达抑制域 (SD)，从 5'- 和 3'- 末端将顺序删除引入 5'-UTR。该缺失实验确定了两个影响报告基因表达的 SD。将这些 SD 插入到 pGL3-Control 载体的 SV40 启动子上游或下游，以观察短 DNA 片段是否可重现抑制效果。我们发现，当 SD 置于启动子下游时，会抑制表达。此外，发现5'-UTR在其5'端区域携带启动子活性。较少的