THE EXPRESSION OF ANGIOGENIN IN ORAL CANCER AND ITS FUNCTIONS

血管生成素在口腔癌中的表达及其功能

• 批准号: 16591999
• 负责人: KISHIMOTO KOJI
• 金额: $ 2.11万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16591999/
• 关键词: angiogenin; oral cancer; RNA interference; angiogenic factor; angiogenesis; ribosomal RNA; cell proliferation; invasion・metastasis; リポゾームRNA

Objectives : Angiogenin is an angiogenic protein that undergoes nuclear translocation in endothelial cells where it accumulates in the nucleolus and stimulates ribosomal RNA transcription, a rate-limiting step in ribosome biogenesis, protein translation, and cell growth. Recently, besides its angiogenic activity, it is revealed that angiogenin also plays a role in cancer cell proliferation through ribosomal RNA transcription. In the current study, we examined the expression of angiogenin in oral cancer and its functions by RNA interference (RNAi).Methods : 1. The expression of angiogenin in biopsy specimens obtained from patients with oral squamous cell carcinoma was examined by immunohistochemistry. 2. Secreted levels of angiogenin and VEGF in various oral cancer cell lines were measured by ELISA. HSC-2 cell that secreted high levels of angiogenin and VEGF was transfected with angiogenin RNAi construct. Then, stable angiogenin RNAi transfectants were selected by puromycin resistance. 3. Vector control and angiogenin RNAi transfectants (RNAi-2, RNAi-3), 1X10^6 cells per mouse, were subcutaneously injected into each mouse. 4. Mice were sacrificed on day 15, and the tumor tissues were formalin-fixed, paraffin-embedded and examined by immunohistochemistry.Results : 1. The expression of angiogenin in oral squamous cell carcinomas was observed in the nuclei or cytoplasms, but not in normal epithelia. 2. Secreted levels of angiogenin and VEGF in various oral cancer cell lines had a wide range of their secretions, and HSC-2 cell secreted high levels of angiogenin and VEGF. 3. Angiogenin RNAi HSC-2 transfectants have decreased tumor volume in athymic mice. 4. Angiogenesis in the tumor tissues of angiogenin RNAi HSC-2 transfectants was inhibited in athymic mice, and moreover, the expression of PCNA also decreased.Conclusion : Angiogenin may play a key role in not only tumor angiogenesis but also cell proliferation in oral cancer.

目的：血管生成素是一种血管生成蛋白，其在内皮细胞中经历核转位，在那里其积聚在核仁中并刺激核糖体RNA转录，这是核糖体生物合成、蛋白质翻译和细胞生长中的限速步骤。最近，除了其血管生成活性外，血管生成素还通过核糖体RNA转录在癌细胞增殖中起作用。本研究应用RNA干扰技术检测血管生成素在口腔癌组织中的表达及其功能。应用免疫组化方法检测口腔鳞癌组织中血管生成素的表达。2.用ELISA法测定不同口腔癌细胞系血管生成素和VEGF的分泌水平。用血管生成素RNAi构建体转染分泌高水平血管生成素和VEGF的HSC-2细胞。然后，通过嘌呤霉素抗性筛选稳定的血管生成素RNAi转染子。3.将载体对照和血管生成素RNAi转染子（RNAi-2、RNAi-3）（每只小鼠1 X 10^6个细胞）皮下注射到每只小鼠中。4.第15天处死小鼠，取肿瘤组织，福尔马林固定，石蜡包埋，免疫组化染色。血管生成素在口腔鳞状细胞癌中主要表达于细胞核或胞浆，而在正常上皮中未见表达。2.不同口腔癌细胞株血管生成素和VEGF的分泌水平差异较大，HSC-2细胞分泌的血管生成素和VEGF水平较高。3.血管生成素RNAi HSC-2转染子减少了无胸腺小鼠的肿瘤体积。4.结论：Angiogenin在口腔癌的血管生成和细胞增殖中起重要作用。