Lin J Q, Xu Z S, Zhang H Y, Yang Z W, Xu Y H
Shanghai Institute of Cell Biology, Academia Sinica.
Shi Yan Sheng Wu Xue Bao. 1995 Sep;28(3):241-6.
We have previously reported that the malignant phenotype of human liver carcinoma cell line BEL-7404 was reversed by antisense EGFR RNA. The aim of this paper is to explore the effects of an oligomer targeted to mRNA for EGFR and growth of BEL-7404 cells. A 21-mer oligodeoxynucleotides (ODNs) complementary to the 5' initiation region of mRNA for EGFR was synthesized and added to medium. The results showed that the growth of BEL-7404 cells was inhibited by ODNs at concentration of 3.2 mumol/L. Inhibition of DNA synthesis of BEL-7404 cells was dose-dependent and reached to 62.1% at 3.2 mumol/L as measured by 3H-thymidine incorporation test. The inhibition of EGFR gene transcription of the cells was up to 10.5% and 14.3% respectively after incubation with ODNs by 5 and 24 hours as measured by densitometric scanning of dot (RNA) blots of EGFR. The EGFR protein (P 170) expression was also found to be blocked by 4 days' antisense oligomer treatment up to 37.4% as measured by densitometric scanning of specific band of Western blot. The oligonucleotide phosphorothioate (S-ODNs) complementary to the same region of the gene was also synthesized and its growth inhibition effects on BEL-7404 cells were compared with those of unmodified oligomers. ODNs attained their highest effect within 30 hours. The proliferation inhibition rate of the cells didn't increase when cells were cultured in serum free medium. In contrast, the S-ODNs induced inhibition reached comparable level after 96 hours treatment as measured by 3H-thymidine uptake and the effect lasted longer, 1 mumol/L S-ODNs showed a little effect on BEL-7404 cells' proliferation. We concluded that the antisense oligomers directed to mRNA for EG-FR could inhibit the BEL-7404 cells growth by blocking the EGFR gene expression in some degree and the phosphorothioate analogues were more stable than the unmodified ODNs in vitro.
我们之前报道过，反义表皮生长因子受体（EGFR）RNA可逆转人肝癌细胞系BEL - 7404的恶性表型。本文旨在探讨针对EGFR mRNA的寡聚体对BEL - 7404细胞生长的影响。合成了一种与EGFR mRNA的5'起始区域互补的21聚体寡脱氧核苷酸（ODN），并将其添加到培养基中。结果显示，浓度为3.2 μmol/L的ODN可抑制BEL - 7404细胞的生长。通过³H - 胸腺嘧啶掺入试验测定，BEL - 7404细胞DNA合成的抑制呈剂量依赖性，在3.2 μmol/L时达到62.1%。通过对EGFR的斑点（RNA）印迹进行光密度扫描测定，与ODN孵育5小时和24小时后，细胞EGFR基因转录的抑制分别高达10.5%和14.3%。通过对蛋白质印迹特异性条带进行光密度扫描测定，还发现经过4天的反义寡聚体处理后，EGFR蛋白（P170）表达被阻断达37.4%。还合成了与该基因相同区域互补的硫代磷酸寡核苷酸（S - ODN），并将其对BEL - 7404细胞的生长抑制作用与未修饰的寡聚体进行了比较。ODN在30小时内达到其最高效果。当细胞在无血清培养基中培养时，细胞的增殖抑制率没有增加。相反，通过³H - 胸腺嘧啶摄取测定，S - ODN诱导的抑制在96小时处理后达到相当水平，且效果持续更长时间，1 μmol/L的S - ODN对BEL - 7404细胞的增殖显示出轻微影响。我们得出结论，针对EG - FR mRNA的反义寡聚体可通过在一定程度上阻断EGFR基因表达来抑制BEL - 7404细胞生长，并且硫代磷酸类似物在体外比未修饰的ODN更稳定。
