转录因子环磷酸腺苷反应元件结合蛋白在紫杉醇诱导HeLa 细胞周期阻滞中的作用及其机制

doi:10.3969/j.issn.0529-1356.2014.04.009

解剖学报 ›› 2014, Vol. 45 ›› Issue (4): 485-492.doi: 10.3969/j.issn.0529-1356.2014.04.009

转录因子环磷酸腺苷反应元件结合蛋白在紫杉醇诱导HeLa 细胞周期阻滞中的作用及其机制

黄帅帅王雪庄海慧王宇多周细武王萍*

宁波大学医学院解剖学与组织学胚胎学系，转化医学重点实验室，浙江宁波 315211

收稿日期:2013-12-26 修回日期:2014-02-25 出版日期:2014-08-06 发布日期:2014-08-06
通讯作者: 王萍 E-mail:wangping2@nbu.edu.cn
基金资助:
细胞骨架结合蛋白通过转录因子CREB调控肿瘤发生发展的分子机制研究. 国家自然科学基金（主持）;转录因子CREB介导的细胞凋亡调控网络在宫颈上皮癌发生发展中的作用及其靶位治疗的研究. 国家自然科学基金（主持）;RhoA介导的细胞骨架调控网络在肿瘤细胞凋亡过程中的作用及其分子机制研究. 浙江省自然科学基金（主持）

Effects of transcription factor cAMP response element binding protein on taxolinduced HeLa cell-cycle arrest

HUANG Shuai-shuai WANG Xue ZHUANG Hai-hui WANG Yu-duo ZHOU Xi-wu WANG Ping*

Department of Anatomy Histology and Embryology, Key Laboratory for Translational Medicine, Ningbo University School of Medicine, Zhejiang Ningbo 315211, China

Received:2013-12-26 Revised:2014-02-25 Online:2014-08-06 Published:2014-08-06
Contact: WANG Ping E-mail:wangping2@nbu.edu.cn

摘要/Abstract

摘要：

目的探讨转录因子环磷酸腺苷反应元件结合蛋白（CREB）在紫杉醇诱导宫颈腺癌HeLa细胞周期阻滞中的作用及其分子机制。方法 MTT法确定紫杉醇的最佳浓度和处理时间；PCR技术构建pCI neo/CREB（PN）重组质粒及pCI neo/CREB-M（PM）定点突变质粒；流式细胞术检测细胞周期，Western blotting检测磷酸化CREB（pCREB）、CREB、cyclins以及CDKs蛋白表达。结果紫杉醇抑制HeLa细胞增殖的有效条件为0.1μmol/L处理24 h。0.1μmol/L紫杉醇诱导G₂/M细胞数量增加，并呈时间依赖性；cyclin A表达量下调，cyclin B1、D1和pCREB表达量上调。此外，紫杉醇的处理对cyclin E、CDK1、CDK2、CDK4和CREB表达量并无显著性改变。然而，PM联合紫杉醇处理后显著性地反转了紫杉醇单独处理引起的cyclin A下调、cyclin B1和cyclin D1的上调，并且细胞周期G₂/M期阻滞显著性减少。结论转录因子CREB介导的靶向细胞周期蛋白表达在紫杉醇诱导的细胞周期阻滞过程中扮演重要角色。

关键词: 宫颈癌, 环磷酸腺苷反应元件结合蛋白, 细胞周期, 流式细胞术, 免疫印迹法

Abstract:

Objective To explore the effects of cAMP response element binding protein (CREB) on taxol-induced cell cycle arrest in HeLa cells. Methods MTT assay was used to determine the optimal concentration and treatment time. PCR method was used to construct the recombinant plasmid pCI neo/CREB（PN）and site-directed mutagenesis recombinant plasmid pCI neo/CREB-M（PM）. Cell cycle was assayed by flow cytometry. Expressions of pCREB, CREB, cyclins and CDKs were assayed by Western blotting. Results The effective conditions of taxol treatment on HeLa cells were 0.1μmol/L for 24 hours. After cells were treated with 0.1μmol/L taxol, G2/M phase was arrested in a time-dependent manner, accomplished with the decrease of cyclin A, a significant increase of cyclin B1, D1 and phosphorylated CREB(pCREB) protein expression, whereas, no marked changes were observed in cyclin E, CDK1, CDK2, CDK4 and CREB expressions. However, combinantion of PM and taxol treatment significantly reduced taxol-induced G₂/M phase arrest, and reversed the effect of taxol-decreased cyclin A, increased cyclin B1 and D1 expression. Conclusion Tanscription factor CREB-mediated specific cyclins play a pivotal role in taxol-induced G₂/M arrest in HeLa cells.

Key words: Cervical cancer, Response element binding protein, Cell cycle, Flow cytometry, Western blotting

黄帅帅王雪庄海慧王宇多周细武王萍*. 转录因子环磷酸腺苷反应元件结合蛋白在紫杉醇诱导HeLa 细胞周期阻滞中的作用及其机制[J]. 解剖学报, 2014, 45(4): 485-492.

HUANG Shuai-shuai WANG Xue ZHUANG Hai-hui WANG Yu-duo ZHOU Xi-wu WANG Ping*. Effects of transcription factor cAMP response element binding protein on taxolinduced HeLa cell-cycle arrest[J]. AAS, 2014, 45(4): 485-492.

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转录因子环磷酸腺苷反应元件结合蛋白在紫杉醇诱导HeLa 细胞周期阻滞中的作用及其机制

Effects of transcription factor cAMP response element binding protein on taxolinduced HeLa cell-cycle arrest

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