THE EXPRESSION OF ANGIOGENIN IN ORAL CANCER AND ITS FUNCTIONS

目的：血管生成素是一种血管生成蛋白，在内皮细胞中发生核易位，在核仁中积累并刺激核糖体RNA转录，这是核糖体生物发生、蛋白质翻译和细胞生长的限速步骤。近年来，血管生成素除了具有血管生成活性外，还通过核糖体RNA转录在癌细胞增殖中发挥作用。在本研究中，我们采用RNA干扰（RNAi）技术检测了血管生成素在口腔癌组织中的表达及其功能。方法：1；应用免疫组织化学方法检测口腔鳞状细胞癌活检标本中血管生成素的表达。2. 采用ELISA法检测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构建物转染分泌高水平血管生成素和VEGF的HSC-2细胞。然后通过对嘌呤霉素的耐药筛选稳定的血管生成素RNAi转染物。3. 每只小鼠皮下注射载体对照和血管生成素RNAi转染物（RNAi-2、RNAi-3），每只小鼠皮下注射1X10^6个细胞。4. 第15天处死小鼠，肿瘤组织经福尔马林固定、石蜡包埋、免疫组化检查。结果：1；血管生成素在口腔鳞状细胞癌的细胞核和细胞质中有表达，而在正常上皮中无表达。2. 各种口腔癌细胞系的血管生成素和VEGF的分泌水平范围广，HSC-2细胞的血管生成素和VEGF的分泌水平较高。3. 血管生成素RNAi HSC-2转染可降低胸腺小鼠的肿瘤体积。4. 血管生成素RNAi HSC-2转染后，胸腺小鼠肿瘤组织中的血管生成受到抑制，PCNA的表达也下降。结论：血管生成素可能在口腔癌肿瘤血管生成和细胞增殖中起关键作用。

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.