RNA干扰MDM2下调人乳腺癌细胞中VEGF的合成并抑制肿瘤血管的生成

目的 探讨小干扰RNA（small interfering RNA, siRNA）抑制人乳腺癌细胞中鼠双微粒体2 （murine double minute 2, MDM2）的表达对癌细胞中血管内皮生长因子（vascular endothelial growth factor, VEGF）合成以及裸鼠移植瘤组织内血管生成的影响。方法 根据MDM2已知的cDNA序列设计并转录合成特异性siRNA，转染高表达MDM2的人乳腺癌MDA-MB-468细胞，低氧培养后应用蛋白质印迹法和ELISA法检测肿瘤细胞及其上清液中VEGF的水平。构建裸鼠乳腺癌移植瘤模型，观察MDM2沉默后肿瘤生长情况，蛋白质印迹法、免疫组织化学及ELISA方法检测荷瘤组织和裸鼠血清标本中的VEGF含量，并以CD34标记血管内皮细胞计数微血管密度（microvessel density, MVD）。结果 siRNA抑制MDM2表达后，MDA-MB-468细胞分泌的VEGF蛋白显著减少（P=0.006）。裸鼠移植瘤模型显示，封闭MDM2表达使荷瘤组织生长变慢（P=0.008），且荷瘤小鼠血清VEGF水平明显减低（P=0.008），荷瘤组织内MVD也明显降低（P=0.003）。结论 MDM2 siRNA能有效减低乳腺癌细胞中VEGF的合成，并抑制裸鼠移植瘤组织内新生血管的生成，为MDM2/VEGF途径抗肿瘤血管生成作用的研究及靶向药物开发提供了新的思路。

MDM2 siRNA Inhibits Angiogenesis by Reducing Vascular Endothelial Growth Factor Production in Breast Cancer Cells

Objective To investigate the inhibitory effects of murine double minute 2 (MDM2) small interfering RNA (siRNA) on vascular endothelial growth factor(VEGF) production in human breast cancer cells and VEGF-mediated angiogenesis in xenograft tissues. Methods siRNA sequences of MDM2 were chemically synthesized and then transfected into human breast cancer cell line MDA-MB-468 which overexpressed MDM2. After transfection, the cells were cultured under hypoxic condition for different period of time. VEGF expression in tumor cells was measured by Western blot. The level of VEGF protein secreted in the culture supernatant was quantified by enzyme-linked immunosorbent assay (ELISA). Further, the stably-transfected cells were transplanted into the nude mice. The tumor growth was analyzed. MDM2 protein expression of tumors was determined by Western blot. Serum levels of VEGF were also measured by ELISA. VEGF expression and microvessel density (MVD) were studied by immunohistochemistry. Results In MDA-MB-468 cells, gene silencing of MDM2 resulted in the down-regulation of VEGF secretion (P=0.006). In vivo, tumorigenicity assay showed that MDM2 depletion restrained tumor growth (P=0.008). Serum levels of VEGF were substantially decreased (P=0.008). MVD in transplanted tumor tissue was also significantly reduced (P=0.003). Conclusion MDM2 knockdown by siRNA causes a strong reduction of VEGF production in breast cancer cells and inhibition of angiogenesis in tumor transplants. The MDM2/VEGF pathway may open a new approach for anti-tumor angiogenesis and molecular targeted drugs development.