Carcinogenesis, Teratogenesis & Mutagenesis ›› 2018, Vol. 30 ›› Issue (2): 126-131.doi: 10.3969/j.issn.1004-616x.2018.02.009

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

PENG Peng1,2, WANG Chaogang2, XU Xinyun1, XIA Junjie1, HUANG Haiyan1, WANG Li3, HUANG Yiqin1, YANG Chen1   

  1. 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

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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

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