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目的研究双调蛋白(AR)反义RNA表达对乳腺癌血管生成的抑制作用。方法乳腺癌NS2T2A1细胞经AR反义cDNA质粒转染后,经筛选获得表达AR反义RNA的AR-AS1及AR-AS3克隆,转染空载体获得NS2T2A1V对照细胞,接种裸鼠皮下形成肿瘤。研究条件培养液对人微血管内皮细胞(HMEC)增殖的影响,以ELISA法测定细胞血管内皮生长因子(VEGF)分泌量。以定量RT-PCR分析肿瘤组织的VEGF表达水平。以免疫组化法标记CD31研究肿瘤内血管数量。结果HMEC在AR-AS1和AR-AS3细胞条件培养液中的增殖比例明显降低。AR-AS1和AR-AS3细胞VEGF分泌量亦降低。AR-AS1和AR-AS3肿瘤的血管数量仅有对照组的50%左右,且VEGF表达显著降低。结论AR反义RNA表达可有效抑制乳腺癌的血管生成,应作为新治疗靶点进一步研究。
Objective To study the inhibitory effect of anti-sense RNA (AR) antisense RNA on angiogenesis in breast cancer. Methods After transfection of NS2T2A1 cells with AR antisense cDNA plasmid, AR-AS1 and AR-AS3 clones expressing AR antisense RNA were screened and transfected into empty vector to obtain NS2T2A1V control cells. Nude mice were inoculated subcutaneously to form tumors. The effects of conditioned media on the proliferation of human microvascular endothelial cells (HMECs) were studied. The secretion of vascular endothelial growth factor (VEGF) was measured by ELISA. Tumor tissues were analyzed for VEGF expression by quantitative RT-PCR. CD31 was used to detect the number of blood vessels in the tumor by immunohistochemistry. Results The proliferation of HMEC in AR-AS1 and AR-AS3 cells was significantly decreased. The amount of VEGF secreted by AR-AS1 and AR-AS3 cells also decreased. The number of vessels in AR-AS1 and AR-AS3 tumors was only about 50% of the control group, and the VEGF expression was significantly reduced. Conclusion AR antisense RNA expression can effectively inhibit angiogenesis in breast cancer and should be further investigated as a new therapeutic target.