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给子细胞带来染色体的不均匀分布，导致非整倍性，并与癌症的进展有关。除了mRNA量的增加外，过度产生极光-A的癌细胞还表达正常细胞中没有的更大的转录物。大的极光-A mRNA亚型具有较长的5 '-非翻译区（5'-UTR），并且来自非编码外显子的替代使用。本研究通过分析5 '-UTR对启动子活性的影响，探讨癌细胞中激酶亚型的表达与激酶过量产生之间的可能关系，将PCR扩增的5'-UTR片段插入pGL 3-Basic载体中的萤火虫荧光素酶基因和phRL-CMV载体中的海肾荧光素酶基因之间，构建双顺反子报告质粒。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 ...更多信息 （IRES）。虽然结果显示在5 '-UTR中不存在IERS，但我们发现5'-UTR片段强烈下调第一个基因的表达。（上游）顺反子。第二个顺反子的下调程度为了简化系统，我们将5 '-UTR片段插入pGL 3-Control载体，位于SV 40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片段下调报告基因表达，证实了5'-UTR中存在负调控元件。为了确定表达抑制结构域（SD），从5 '-和3'-末端向5 '-UTR中引入连续缺失。该缺失实验鉴定了影响报告基因表达的两个SD。将这些SD插入到pGL 3-对照载体的SV 40启动子上游或下游，以观察用短DNA片段是否可再现抑制效果。我们发现，当放置在启动子下游时，SDs抑制表达。此外，发现5 '-UTR在其5'-末端区域携带启动子活性。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