沉默CDK7基因对HepG2细胞株的抗增殖作用

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (01): 58-61.

沉默CDK7基因对HepG2细胞株的抗增殖作用

赵爱国, 吴曙光

南方医科大学药物研究所, 广东广州510515

出版日期:2005-01-20 发布日期:2005-01-20
基金资助:
收稿日期:2004-3-10。
作者简介:赵爱国(1970- ),男,广州中医药大学博士后,电话:020-61648167
通讯作者:吴曙光,男,博士研究生导师,电话:020-61648167

Effect of CDK7 gene silencing against hepatoblastoma HepG2 cell proliferation

ZHAO Ai-guo, WU Shu-guang

南方医科大学药物研究所, 广东广州510515

Online:2005-01-20 Published:2005-01-20

摘要/Abstract

摘要： 目的筛选对体外培养HepG2细胞抗增殖作用最强的反义寡脱氧核苷酸(anti-senseoligodeoxynucleotides,ASODN)序列,探讨经二次优化后该ASODN片段的抗增殖作用。方法根据RNAStructure3.71计算机软件模拟人CDK7mRNA获得的二级结构确定待选ASODN,经OligoWalk程序分析这些片段与mRNA互补时的热力学参数变化,以此优选待筛ASODN,以体外培养人肝癌HepG2细胞为筛选体系获得抗增殖作用最强的ASODN,对该片段分别依次向互补区上、下游错位1~5个碱基获得10条ASODN,经结构优化后进行二次筛选。结果初筛结果显示互补于人CDK7mRNA第284~303片段的部分硫代修饰ASODN抗增殖作用最强,为(40.4±12.6)%;二次筛选显示互补于第287~306片段的全磷酸硫代修饰ASODN抗增殖作用最强,抑制率为(68.3±2.6)%,半数抑制浓度为(51.9±8.6)nmol/L。结论筛选得到的ASODN片段可显著抑制体外培养HepG2细胞的增殖,有可能作为先导化合物开发特异性CDK7抑制剂。

Abstract: Objective To screen the antisense oligodeoxynucleotides (ASODN) that inhibit cultured hepatoblastoma HepG2 cell proliferation, and evaluate the antiproliferative potency of modified ASODN. Methods ASODN sequences were selected based on the secondary structure of human CDK7 mRNA predicted with RNAStructure 3.71 software. The binding thermodynamics of CDK7 mRNA to ASODN was analyzed by OligoWalk program. The sequences with the strongest effect against cultured HepG2 cell proliferation in vitro were selected, and the fragments complementary to 1-5 bases upstream or downstream to the complementary region were structurally modified and screened. Results The partial phosphorothioate ASODN complementary to 284-303 region of human CDK7 mRNA was the most powerful inhibitor, and the antiproliferative activity reached 40.4±12.6%; in the second round of screening, the antiproliferative activity of the full phosphorothioate ASODN complementary to the 287-306 region of the mRNA on HepG2 cells was 68.3±2.6%, with IC50 of 51.9±8.6 nmol/L. Conclusion Proliferation of HepG2 cells can be significantly inhibited by the screened ASODN, which might be used as a lead compound in the development of specific CDK7 inhibitors.

中图分类号:

赵爱国, 吴曙光. 沉默CDK7基因对HepG2细胞株的抗增殖作用[J]. 南方医科大学学报, 2005, 25(01): 58-61.

ZHAO Ai-guo, WU Shu-guang. Effect of CDK7 gene silencing against hepatoblastoma HepG2 cell proliferation[J]. Journal of Southern Medical University, 2005, 25(01): 58-61.

参考文献

[1] Kasten MM, Giordano A. pRb and the cdks in apoptosis and the cell cycle[J]. Cell Death Differ, 1998, 5(2): 132-40.
[2] Fisher RP, Morgan DO. CAK in TFIIH: crucial connection or confounding coincidence [J] ? Biochim Biophys Acta, 1996, 1288(1):7-10.
[3] Rossignol M, Kolb-Cheynel I, Egly JM. Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH [J]. EMBO J, 1997, 16(7): 1628-37.
[4] 宋海峰,汤仲明.反义药物作用中的"靶二级结构域"[J].药学学报,2001,36(8):585-9.Song HF, Tang ZM. "Target secondary structural motif′ in the action of antisense oligodeoxynucleotides [J]. Acta Pharmaceutica Sin, 2001, 36(8): 585-9.
[5] Sugimoto N, Yasumatsu I. A new concept for the design of antisense oligonucleotides based on nucleic acid thermostability [J]. Curr Med Chem Anti-Canc Agents, 2001, 1(1): 95-112.
[6] Jaaskelainen I, Honkakoski P, Urtti A. In vitro delivery ofantisense oligonucleotides [J]. Cell Mol Biol Lett, 2002, 7(2): 236-7.
[7] Bochar DA, Pan ZQ, Knights R, et al. Inhibition of transcription by the trimeric cyclin-dependent kinase 7 complex [J]. J Biol Chem,1999, 274(19): 13162-6.
[8] Song HF, Tang ZM, Yuan SJ, et al. Application of secondary structure prediction in antisense drug design targeting protein kinase C-alpha mRNA and QSAR analysis [J]. Acta Pharmacol Sin, 2000,21(1): 80-6.
[9] 孔伟东,李彦豪,曾庆乐,等.平阳霉素对血管内皮细胞的生长抑制作用和细胞周期的影响[J].第一军医大学学报,2003,23(8):830-2.Kong WD, Li YH, Zeng QL, et al. Effect of pingyangmycin on the growth and cell cycle of human vascular endothelial cells [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2003, 23(8): 830-2.
[10] Peter MF, Graham B, Carol M, et al. Cell cycle target validation: approaches and successes[J]. Targets, 2003, 2(4): 154-61.
[11] 陈汉源.细胞周期素D1与癌症[J].第一军医大学学报(J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao), 1999, 19(6): 586-91.
[12] Dyson N. The regulation of E2F by pRB-family proteins[J]. Genes Dev, 1998, 12(15): 2245-62.
[13] Knockaert M, Greengard P, Meijer L. Pharmacological inhibitors of cyclin-dependent kinases[J]. Trends Pharmacol Sci, 2002, 23(9):417-25.
[14] Senderowicz AM, Sausville EA. Preclinical and clinical development of cyclin-dependent kinase modulators [J]. J Natl Cancer Inst,2000, 92(5): 376-87.
[15] Zhao B, Bower MJ, McDevitt PJ, et al. Structural basis for Chkl inhibition by UCN-01 [J]. J Biol Chem, 2002, 277(48): 46609-15.
[16] Sausville EA. Complexities in the development of cyclin-dependent kinase inhibitor drugs [J]. Trends Mol Med, 2002, 8 (4 Suppl):S32-7.