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目的筛选出可与血管内皮生长因子受体2(KDR)mRNA高效、特异结合的反义寡核苷酸,探讨其体外抗肿瘤作用。方法先用寡核苷酸库杂交和计算机预测初筛反义寡核苷酸;经寡核苷酸芯片杂交验证,挑选与KDR mRNA有强杂交信号的反义寡核苷酸,用MTT法、逆转录聚合酶链反应(RT-PCR)和免疫印迹(Western blotting)探讨反义寡核苷酸对乳腺癌细胞系MCF-7细胞增殖和KDR表达的抑制作用。结果在寡核苷酸库杂交筛选出的13条反义寡核苷酸中,有8条(8/13,61.5%)在芯片杂交中显示较强杂交信号;而在计算机预测设计的17条探针中,只有1条显示较强信号。合成杂交信号较强的9条硫代反义寡核苷酸,均能有效抑制MCF-7细胞增殖,并呈剂量依赖性。其中抑制率最高的2条反义寡核苷酸为asON4和asON7,由寡核苷酸库杂交联合芯片杂交筛出,在0.8μmol/L时抑制率分别为51.6%和62.2%;同时,这2条反义寡核苷酸在mRNA水平和蛋白质水平抑制了KDR基因表达,并呈剂量相关性。结论寡核苷酸库杂交筛选和寡核苷酸芯片杂交筛选的结果有较好一致性,将寡核苷酸库和寡核苷酸芯片联用是一种较好筛选反义寡核苷酸方法。KDR反义寡核苷酸有明显的抗肿瘤作用。
Objective To screen efficient and specific antisense oligodeoxynucleotides that bind to vascular endothelial growth factor receptor 2 (KDR) mRNA and to explore its antitumor activity in vitro. Methods Antisense oligodeoxynucleotides were first predicted by oligonucleotide library hybridization and computerized prediction. Antisense oligonucleotides with strong hybridization signal with KDR mRNA were selected by oligonucleotide hybridization. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to investigate the inhibitory effect of antisense oligonucleotide on the proliferation and KDR expression of breast cancer cell line MCF-7. Results Of the 13 antisense oligonucleotides screened by oligonucleotide library hybridization, 8 (8/13, 61.5%) of the 13 antisense oligonucleotides displayed strong hybridization signals in the hybridization of chips. In computer-aided design, 17 Only 1 of the probes shows a stronger signal. Nine synthetic antisense oligonucleotides with strong hybridization signal could inhibit the proliferation of MCF-7 cells in a dose-dependent manner. The two antisense oligonucleotides with the highest inhibition rates were asON4 and asON7, which were screened by oligonucleotide hybridization hybridization and chip hybridization. The inhibitory rates were 51.6% and 62.2% at 0.8μmol / L, respectively. At the same time, Two antisense oligonucleotides inhibited KDR gene expression at mRNA and protein levels in a dose-dependent manner. Conclusion The result of oligonucleotide hybridization screening and oligonucleotide hybridization screening showed good agreement. The combination of oligonucleotide pool and oligonucleotide chip is a better method for screening antisense oligonucleotides method. KDR antisense oligonucleotide has obvious anti-tumor effect.