StAR基因高表达对DEHP致MCF-7细胞凋亡作用的影响

doi:10.3969/j.issn.1004-616x.2018.02.009

癌变·畸变·突变 ›› 2018, Vol. 30 ›› Issue (2): 126-131.doi: 10.3969/j.issn.1004-616x.2018.02.009

StAR基因高表达对DEHP致MCF-7细胞凋亡作用的影响

彭鹏^1,2, 王潮岗², 徐新云¹, 夏俊杰¹, 黄海燕¹, 王利³, 黄奕钦¹, 杨晨¹

1. 深圳市疾病预防控制中心毒理研究所, 广东深圳 518055;
2. 深圳大学生命与海洋科学学院, 广东深圳 518060;
3. 南华大学公共卫生学院, 湖南衡阳 421001

收稿日期:2017-08-17 修回日期:2018-03-15 出版日期:2018-03-30 发布日期:2018-03-30
通讯作者: 徐新云,E-mail:xyxu2008@163.com E-mail:xyxu2008@163.com
作者简介:彭鹏,E-mail:843709278@qq.com
基金资助:
深圳市科技研发基础研究项目（JCYJ20170306160553495）

Construction cells over-expressing the StAR gene and its apoptotic response to DEHP

PENG Peng^1,2, WANG Chaogang², XU Xinyun¹, XIA Junjie¹, HUANG Haiyan¹, WANG Li³, HUANG Yiqin¹, YANG Chen¹

1. Institue of Toxicology, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, Guangdong;
2. School of Life Science, Shenzhen University, Shenzhen 518060, Guangdong;
3. School of Public Health, South China University, Hengyang 421001, Hunan, China

Received:2017-08-17 Revised:2018-03-15 Online:2018-03-30 Published:2018-03-30

摘要/Abstract

摘要： 目的：构建类固醇合成急性调节蛋白（StAR）基因高表达载体，建立StAR基因高表达细胞，研究StAR基因高表达对邻苯二甲酸二-（2-乙基己）酯（DEHP）毒性作用的影响。方法：根据GenBank提供的StAR基因cDNA序列设计引物，PCR扩增StAR基因并将其克隆到慢病毒载体中，构建StAR基因高表达MCF-7细胞，通过基因测序、荧光定量PCR和Western blot鉴定细胞。用不同剂量DEHP染毒MCF-7细胞和StAR基因高表达MCF-7细胞24 h，检测细胞凋亡相关基因Bax、Caspase-3、Caspase-8在mRNA和蛋白表达水平的变化。结果：基因测序证明构建的StAR基因高表达载体序列正确，荧光定量PCR检测StAR基因转染细胞比正常MCF-7细胞StAR mRNA表达水平升高591.9倍（P < 0.01），Western blot实验结果显示，StAR基因转染细胞比正常MCF-7细胞StAR蛋白表达水平升高190%（P < 0.01）。不同剂量DEHP染毒StAR基因高表达细胞后，荧光定量PCR结果显示，StAR基因高表达细胞中Bax、Caspase-3和Caspase-8 mRNA表达水平较对照组（0 mmol/L DEHP）显著升高，差异均有统计学意义（P < 0.05或P < 0.01）。Western blot实验显示，StAR基因高表达细胞中Bax、Caspase-3和Caspase-8蛋白表达水平比对照组显著升高，差异均有统计学意义（P < 0.05或P < 0.01）。结论：本实验成功构建了StAR基因高表达细胞，DEHP处理后StAR基因高表达细胞的凋亡相关基因表达水平较MCF-7细胞升高，提示StAR基因具有促进DEHP致细胞凋亡作用。

关键词: 邻苯二甲酸二-(2-乙基己)酯, 类固醇合成急性调节蛋白, 基因高表达, 凋亡基因

Abstract: OBJECTIVE: To construct cells with over-expression of the StAR gene and to study its apoptotic response to di-(2-ethylhexyl) phthalate (DEHP). METHODS: Primers were designed according to cDNA sequence of the StAR gene from GenBank. The gene was amplified using PCR and ligated into the lentiviral vector pLVX-CMV-ZSgreen-PGK-Puro. 293FT cells were transfected with the recombinant vector and then MCF-7 cells were transfected. Cells with over-expression of the StAR gene were identified by gene sequencing,real-time quantitative PCR and western blot. Then the StAR gene over-expression cells and MCF-7 cells were treated with various doses of DEHP for 24 h to detect expression of apoptosis genes including Bax,Caspase-3 and Caspase-8. RESULTS: The sequence contained in the recombinant vector was exactly the same as the StAR gene from GenBank. StAR gene expression level in the transfected MCF-7 cells was 591.9 times higher than that of control MCF-7 cells. StAR protein level in the transfected MCF-7 cells was 190% higher than that of control MCF-7 cells. After DEHP treatment of the transfected and control MCF-7 cells,qPCR results show that mRNA levels of Bax,Caspase-3 and Caspase-8 were significantly higher in the transfected MCF-7 cells. Additionally,mRNA levels of Bax,Caspase-3 and Caspase-8 increased significantly (P < 0.05 or P < 0.01) in the transected compared with the similarly treated non-transfected MCF-7 cells. Western blot results show that the protein expression levels of Bax,Caspase-3 and Caspase-8 also increased significantly in the transfected MCF-7 cells. Protein expression levels of Bax,Caspase-3 and Caspase-8 were also increased significantly (P < 0.05 or P < 0.01) except in the 0.2 mmol/L treatment group. CONCLUSION: The StAR gene over-expression cells were successfully constructed. These cells responded to DEHP treatments by showing significant increase in apoptotic gene expression. These findings indicate that StAR gene promoted DEHP induced-apoptosis.

Key words: di-(2-ethylhexyl) phthalate, steroidogenio acute regulatory protein, gene over-expression, apoptosis gene

中图分类号:

R394.6

彭鹏, 王潮岗, 徐新云, 夏俊杰, 黄海燕, 王利, 黄奕钦, 杨晨. StAR基因高表达对DEHP致MCF-7细胞凋亡作用的影响[J]. 癌变·畸变·突变, 2018, 30(2): 126-131.

PENG Peng, WANG Chaogang, XU Xinyun, XIA Junjie, HUANG Haiyan, WANG Li, HUANG Yiqin, YANG Chen. Construction cells over-expressing the StAR gene and its apoptotic response to DEHP[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(2): 126-131.

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StAR基因高表达对DEHP致MCF-7细胞凋亡作用的影响

Construction cells over-expressing the StAR gene and its apoptotic response to DEHP

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