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

2006 Fiscal Year Final Research Report Summary

• Project/Area Number: 17591766
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Obstetrics and gynecology
• Research Institution: Hyogo College of Medicine
• Principal Investigator: MORINAGA Tomonori Hyogo College of Medicine, Faculty of Medicine, Research Associate, 医学部, 助手 (10351818)
• Co-Investigator(Kenkyū-buntansha): HASHIMOTO Tomoko (TAMAOKI Tomoko) Hycgo College of Medicine, Faculty of Medicine, Professor, 医学部, 教授 (10172868) ; TSUJI Yoshiyuki Hycgo College of Medicine, Faculty of Medicine, Part-time teacher, 医学部, 非常勤講師 (60148658) ; YOSHIKAWA Reigetsu Hycgo College of Medicine, Faculty of Medicine, Research Associate, 医学部, 助手 (90319864)
• Project Period (FY): 2005 – 2006
• Keywords: Cell Cycle / M Phase / Translation regulation / Gene Expression / Cancer-related Genes

Research Abstract

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 (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.