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

利用差异显示寻找口腔鳞状细胞癌侵袭/转移相关基因

• 批准号: 11672002
• 负责人: OKUMURA Kazuhiko
• 金额: $ 1.41万
• 依托单位: Health Sciences University of Hokkaido
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672002/
• 关键词: Invasion; Metastasis Related Genes; Differential Display PCR; Oral Squamous Cell Carcinoma Cells

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

我们应用mRNA差异显示技术筛选SAS人舌低分化鳞状细胞癌高、低侵袭克隆细胞差异表达的基因。通过使用荧光差异显示和RT-PCR分析，以确认检测差异表达的cDNA片段的存在下，我们已经分离出两个部分测序的基因，表达在高或低侵袭SAS克隆细胞。材料与方法：使用人舌低分化鳞状细胞癌细胞系SAS衍生的具有高侵袭性的SAS-H1细胞和具有低侵袭性的SAS-L1细胞。测试TAKARA的罗丹明荧光差异显示试剂盒（9种不同的锚和24种任意引物组合）以鉴定高侵袭细胞和低侵袭细胞之间的差异显示mRNA。高、低侵袭克隆细胞差异表达条带的筛选。被选中的乐队是 ...更多信息 从凝胶上洗脱、纯化并再扩增。所有片段在再扩增时均显示单一条带。将cDNA片段克隆到平末端克隆载体中。用ABI PRISM 310基因分析仪对部分cDNA片段进行测序分析。将这些序列输入DNA数据库（BLAST）以检查同源性搜索。结果如下：为了确认mRNA差异显示谱中观察到的LIEG-1和HIEG-1的表达模式，我们使用cDNA片段分离的匹配引物，通过RT-PCR分析检测了这些基因在高侵袭性和低侵袭性SAS克隆细胞中的表达。RT-PCR分析结果与差异显示实验中观察到的表达模式一致。LIEG-1在低侵袭性克隆细胞中表达增加，如SAS-L1。相反，HIEG-1在高侵袭性克隆细胞SAS-H1中表达增加。LIEG-1与1号染色体上的克隆RP 5 - 926 E3的同源性为100%。经计算机检索GenBank/EMBL DNA数据库，未发现HIEG-1基因的同源序列。我们已经确定了两个基因; LIEG-1和HIEG-1。这些基因的全长cDNA克隆正在进行中。少

项目成果

Kinoshita, R., Okumura, K., Hagino, T., Arakawa, T., Okazaki, Y.Takuma, T.and Kanazawa, M.: "Search for Invasion/Metastasis Related Genes of Oral Squamous Cell Carcinoma"Higashi Nippon Dent.J.. 19 (2). 24 (2000)

Kinoshita, R.、Okumura, K.、Hagino, T.、Arakawa, T.、Okazaki, Y.Takuma, T.和 Kanazawa, M.：“寻找口腔鳞状细胞癌的侵袭/转移相关基因”Higashi Nippon

木下隆二,奥村一彦 ら: "口腔扁平上皮癌の浸潤転移関連遺伝子の探索"東日本歯学会雑誌. 19・2. 24 (2000)

Ryuji Kinoshita、Kazuhiko Okumura 等：“寻找与口腔鳞状细胞癌的侵袭和转移相关的基因”《东日本牙科学会杂志》19, 2. 24 (2000)。