[Construction of 3β-HSD gene silencing cell line and its effects on apoptosis induced by DEHP]

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.