Construction of 3β-HSD gene silencing cell line and its effects on apoptosis induced by DEHP
10.3760/cma.j.issn.1001-9391.2019.01.003
- VernacularTitle: 3β-HSD基因沉默细胞株的建立及其对DEHP致凋亡的影响
- Author:
Li WANG
1
,
2
;
Xiaoyun QIN
1
;
Xinyun XU
1
;
Huaicai ZENG
1
,
2
;
Kai ZHENG
3
;
Bingyu WANG
1
,
3
;
Dingxin LONG
3
Author Information
1. Institue of Toxicology, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China
2. The Public Sanitation College of Nanhua University, Hengyang 421001, China
3. The Public Sanitation College of Nanhua University, Hengyang 421001, China
- Publication Type:Journal Article
- Keywords:
3β
-hydroxysteroid dehydrogenase;
Gene silencing;
Di- (2-ethylhexyl) phthalate;
Apoptosis
- From:
Chinese Journal of Industrial Hygiene and Occupational Diseases
2019;37(1):13-19
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct 3β-HSD gene shRNA lentivirus interference vecto, then transfect into human MCF-7 cells, and construct cell line with 3β-HSD gene silencing, finally to study the effects of 3β-HSD on apoptosis induced by di- (2-ethylhexyl) phthalate (DEHP) .
Methods:According to the mRNA sequence of 3β-HSD gene provided by GenBank, three interference sequences were designed and connected to PLVX-shRNA2-puro after annealing. The recombinant lentivirus vector was transfected into 293FT cells, the virus supernatants were collected and infected with MCF-7 cells. After puromycin screening, MCF-7 cells with 3β-HSD gene silencing were constructed. The cells with 3β-HSD gene silencing were identified by real-time quantitative PCR and western blot. Then the 3β-HSD gene silencing cells and MCF-7 cells were treated at various doses of DEHP for 24 hours to detect the gene expression and protein expression of apoptosis genes including Bax, Caspase-3 and Caspase-8.
Results:The interference sequence of 3β-HSD gene inserted into lentivirus vector PLVX-shRNA2-puro is consistent with the designed sequence. 3β-HSD gene expression level in MCF-7 cells with 3β-HSD gene silencing was 77% lower than than that of control MCF-7 cells. 3β-HSD protein level in MCF-7 cells with 3β-HSD gene silencing was 74% lower than that of control MCF-7 cells. After DEHP treatment in MCF-7 cells with 3β-HSD gene silencing and control MCF-7 cells, qRT-PCR results showed that Bax gene expression levels increased by 28%-54%, Caspase-3 gene increased by 13%-49%, Caspase-8 gene increased by 21%-70% in MCF-7 cells when compared with the control group. Additionally, in the 3β-HSD gene silencing cells, Bax gene expression level decreased by 11%-28%, Caspase-3 gene expression decreased by 12%-23%, Caspase-8 gene expression decreased by 11%-34%, compared with the same treatment group of MCF-7 cells. Western blot results showed that Bax protein expression level increased by 28%-61%, Caspase-3 protein expression level increased by 40%-48%, Caspase-8 protein increased by 31%-84% in MCF-7 cells when compared with the control group. In 3β-HSD gene silencing cells, Bax protein expression level increased by 11%-27%, Caspase-3 protein increased by 21%-40%, Caspase-8 protein increased by 12%-25%, compared with the same treatment group of MCF-7 cells.
Conclusion:The stable 3β-HSD gene silencing cell line are successfully constructed in this study. DEHP can induce increased expression of apoptotic gene and protein. Silencing of 3β-HSD gene can inhibit the activation of apoptotic gene by DEHP in a certain degree.
