反向寡脱氧核苷酸抑制细胞周期素D_1表达及对黑素瘤细胞增殖的影响

Antisense Oligodeoxynucleotide Inhibits the Expression of Cyclin D_1 and Its Influence on Growth of Melanoma Cells

目的探讨细胞周期素D1 反向寡脱氧核苷酸对恶性黑素瘤的潜在治疗作用。方法合成针对细胞周期素D1 mRNA翻译起始部位序列的20bp硫代磷酸反向寡脱氧核苷酸(AS ODN)。采用RT PCR检测AS ODN对黑素瘤(B16 细胞)细胞周期素D1 mRNA水平的影响 ;用流式细胞仪检测细胞周期素D1 蛋白表达及细胞周期分布 ;固绿染色计数、集落形成抑制试验体外观察AS ODN对B16 细胞增殖的影响。结果细胞周期素D1 mRNA反向链明显抑制B16 细胞细胞周期素D1 mRNA和细胞周期素D1 蛋白表达 ,降低S期细胞百分比 ,且明显抑制B16 细胞生长和集落形成。结论细胞周期素D1 反向寡脱氧核苷酸能有效抑制黑素瘤细胞增殖。

Objective To explore the therapeutic potential of antisense oligodeoxynucleotide to cyclin D1 for malignant melanoma. Methods Three 20 mers phosphorothioate oligodeoxynucleotides (ODNs) were synthesized: (1)Cyclin D1 antisense ODN (AS ODN) targeting the initiation site of translation in cyclin D1 mRNA;(2)Cyclin D1 sense ODN(S ODN) homologous to the initiation site of translation in cyclin D1 mRNA;(3)Random ODN (R ODN). The level of cyclin D1 mRNA was observed by RT PCR. The level of cyclin D1 protein and the distribution of cell cycle progression were examined by flow cytometry. The inhibitory effect of ODN for the growth of melanoma (B16) cells was observed by solid green staining and colony forming inhibitory test. Results AS ODN inhibited obviously the level of cyclin D1 mRNA expression while S ODN and R ODN did not. The level of cyclin D1 protein was significantly lower in AS ODN treated cells than that in controls (P< 0.01)but not in S ODN and R ODN treated cells(P >0.05). AS ODN inhibited B16 cells entering S phase from G1 phase, however, both S ODN and R ODN had no such effect. Cells treated with AS ODN, S ODN, R ODN displayed a colony forming inhibitory rate of 63.64%, 18.19%, and 31.82%, and a cell growth inhibitory rate of 64.73%, 26.51%, and 29.64%, respectively. Conclusion Cyclin D1 antisense oligodeoxynucleotide has strong inhibitory effect on the growth of melanoma cells.