Search for Invasion/Metastasis Related Genes of Oral Squamous Cell Carcinoma using Differential Display

Research Project

Project/Area Number	11672002
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Surgical dentistry
Research Institution	Health Sciences University of Hokkaido
Principal Investigator	OKUMURA Kazuhiko Health Sciences University of Hokkaido, School of Dentistry, Assistant Professor, 歯学部, 講師 (60194510)
Co-Investigator(Kenkyū-buntansha)	TOMIOKA Keiko Health Sciences University of Hokkaido, School of Dentistry, Research Assistant, 歯学部, 助手 (10227613)
Project Period (FY)	1999 – 2000
Project Status	Completed (Fiscal Year 2000)
Budget Amount *help	¥2,200,000 (Direct Cost: ¥2,200,000) Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000) Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Keywords	Invasion / Metastasis Related Genes / Differential Display PCR / Oral Squamous Cell Carcinoma Cells
Research Abstract	We applied mRNA differential display method to identify genes differentially expressed between high and low-invasion cloned cells derived from SAS human tongue poorly differentiated squamous cell carcinoma were used. By using fluorescence differential display and RT-PCR analysis to confirm the presence of cDNA fragments that detect differential expression, we have isolated two partial sequenced genes that expressed in either high or low-invasion SAS cloned cells. Materials and Methods : A human tongue poorly differentiated squamous cell carcinoma cell line SAS-derived SAS-H1 cells with high invasive property and SAS-L1 cells with low invasive property were used. TAKARA's rhodamine fluorescence differential display kit (nine different anchor and twenty-four arbitrary primer combinations) were tested identify differential displayed mRNAs between high and low invasion cells. The selection of differentially expressed bands between high and low-invasion cloned cells. The selected bands were … More eluted from the gels, purified, and reamplified. All fragments exhibited single bands upon reamplification. The cDNA fragments were cloned into blunt-end cloning vector. Sequencing analysis of partial cDNA fragments was determined using the ABI PRISM 310Genetic Analyzer. These sequences were entered in the DNA database (BLAST) to examine the homology search. Results : To confirm the expression patterns of the LIEG-1 and HIEG-1 observed in the mRNA differential display profile, we examined the expression of those genes in high and low invasion SAS cloned cells by RT-PCR analysis, using the cDNA fragment isolated matched primers. The result of RT-PCR analysis was consistent with the expression pattern observed in the differential display experiment. The LIEG-1 was expressed increase in low invasion cloned cells as SAS-L1. In contrast, the HIEG-1 was increased expression in high invasion cloned cells as SAS-H1. LIEG-1 was identified 100% homologous sequence from clone RP5-926E3 on chromosome 1. HIEG-1 was identified no homologous sequence by computer search against the GenBank/EMBL DNA database. We have identified two genes ; LIEG-1 and HIEG-1. Cloning of full-length cDNAs for these genes is now in progress. Less