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

极光-A激酶的过度表达被认为会导致子代细胞染色体分布不均匀，导致非整倍体，并与癌症的进展有关。除了增加信使核糖核酸的数量外，过量产生极光-A的癌细胞还表达正常细胞中没有的更大的转录本。大的AURORA-A mRNA亚型有更长的5‘-非翻译区(5’-UTR)，来自非编码外显子的替代使用。在本工作中，我们分析了5‘-UTR对启动子活性的影响，以探讨其异构体的表达与肿瘤细胞中蛋白激酶的过量生产之间的可能关系。通过在pGL3-Basic载体的萤火虫荧光素酶基因和phRL-CMV载体的肾状荧光素酶基因之间插入PCR扩增的5’-UTR片段，构建了双顺反子报告载体。将这些质粒导入培养的癌细胞，观察5‘-非编码区片段是否携带内部核糖体进入位点…更多(IRES)。虽然结果显示5‘-UTR中没有IERS，但我们发现5’-UTR片段强烈下调第一(上游)顺反子的表达，下调第二(下游)顺反子的程度取决于所使用的细胞，提示了复杂的调控机制。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之间，以产生单顺反子报告载体。我们发现5‘-UTR片段下调了报告基因的表达，证实了5’-UTR中存在负调控元件。为了确定表达抑制域(SDS)，在5‘-UTR5’-端和3‘-端引入了顺序删除。本缺失实验确定了两个影响报告基因表达的SDS。将这些SDS插入到SV40启动子上游或下游的pGL3-Control载体中，观察短DNA片段是否能重现抑制作用。我们发现，当将十二烷基硫酸钠放在启动子下游时，表达受到抑制。此外，还发现5‘-非编码区在其5’端区域具有启动子活性。较少

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