The Role Of 3?-HSD Gene In Cytotoxicity Of MCF-7 Induced By DEHP

Objective:To study the effect of DEHP on the expression of 3?-HSD in MCF-7 cells,construct 3?-HSD gene silencing cell line and 3?-HSD gene overexpressing cell line,and explore the role of 3?-HSD in the reproductive and endocrine toxicity of DEHP in order to further understand the toxic mechanism of DEHP.Methods:MCF-7 cells were treated with DEHP at a concentration of 0.00-0.80 mmol/L(12-48 h)or treated with a single concentration of 0.80 mmol/L DEHP for different times(3-48h).Then observe the morphological changes of the cells,as well as detect changes in gene and protein expression levels of 3?-HSD.The 3?-HSD gene silencing vector was constructed by RNA interference technology.The 3?-HSD gene silencing vector was transfected into MCF-7 cells to construct a 3?-HSD gene silencing cell line.The 3?-HSD gene overexpression vector was constructed by molecular cloning.The 3?-HSD gene overexpression vector was transfected into MCF-7 cells to construct 3?-HSD gene overexpressing cell line.The effects of RNA interference and gene expression were identified by gene sequencing,quantitative PCR and Western blot.To investigate the effect of silencing or overexpression of 3?-HSD on the toxicity of DEHP.MCF-7 cells were used as control,and 3?-HSD silencing cells and overexpressing cells were treated with DEHP at a concentration of 0.00-0.80 mmol/L for 24 hours.Morphological changes were observed.Changes in oxidative damage markers such as MDA,SOD,GSH and GSHPX in three types of cells were detected.Q-PCR detection of DNA damage repair gene(h OGG1,h MTH1),apoptotic gene(Bax,Caspase-3,Caspase-8)and estrogen receptor gene(ER?,ER?)m RNA expression levels,Western blot detection of the corresponding protein expression levels.Signaling pathways and proteomics studies were conducted.Three cells were exposed to DEHP at a concentration of 0.00-0.40 mmol/L for 24 h.Changes in the expression levels of signaling pathway related genes(p38,ERK1,ERK2,i NOS,PKC?)were detected in cells.After treatment with signal pathway inhibitors,DEHP was used to detect changes in the expression levels of signaling pathway related genes and apoptotic genes in MCF-7 cells.Quantitative proteomic analysis was performed on three types of cells treated with 0.40 mmol/L DEHP for 24 h to screen for differential proteins.Results:The levels of 3?-HSD gene expression were increased in MCF-7 cells exposed to different concentrations of DEHP for 12 to 48 h,and the expression levels of 3?-HSD were higher than those in the control group.As the dose increased,they showed an upward trend.Different doses of DEHP treated cells 24 h,the 3?-HSD protein expression level also showed an upward trend with increasing dose.At a single concentration of 0.80 mmol/L DEHP,the expression level of 3?-HSD m RNA was higher than that of the control group,and it increased with the prolonged exposure time.The interference sequence in the recombinant PLVX-HSD-sh RNA recombinant vector constructed by gene sequencing was consistent with the designed sh RNA sequence,and the 3?-HSD gene sequence in the gene high expression vector p LVX-HSD was identical to the sequence in Gen Bank.Q-PCR showed that the expression level of 3?-HSD gene in the silenced cells was reduced by 77% compared with the control group MCF-7 cells,and the 3?-HSD gene expression level in the overexpressed cells was 105 times higher than that in the control group.Western blot showed that the level of 3?-HSD protein in the silenced cells X was decreased by 74% compared with the control group MCF-7 cells,and the 3?-HSD protein in the overexpressed cells was 80% higher than that in the control group.Different concentrations of DEHP treated cells for 24 hours,with the increase of DEHP exposure dose,MDA content in MCF-7 cells increased,SOD activity,GSH content and GSH-PX activity decreased.The expressions of h OGG1,h MTH1,Bax,Caspase-3,Caspase-8,ERa,and ER? in the gene and protein were higher than those in the control group.Compared with the same dose of MCF-7 cells,MDA content in 3?-HSD silencing cells was increased,SOD activity,GSH content and GSH-PX activity were significantly decreased.The expression of h OGG1,h MTH1,Bax,Caspase-3,Caspase-8,ER? and ER? decreased in both gene and protein expression.Compared with the same dose of MCF-7 cells,the content of MDA in 3?-HSD overexpressing cells was decreased,SOD activity,GSH content,and GSH-PX activity were significantly increased.The expression of h OGG1,h MTH1,Bax,Caspase-3,Caspase-8,ER? and ER? increased in gene and protein expression.After treatment with p38 inhibitors,the expression of p38,i NOS,Bax,Caspase-3,and Caspase-8 was decreased in DEHP treated group;after treatment with ERK1/2 inhibitor,the expression of ERK1 and ERK2 was decreased in DEHP treated group;after treatment with i NOS inhibitor,the expression of i NOS,Bax,Caspase-3,and Caspase-8 was decreased in DEHP treated group,and the expression of PKC? in DEHP treated group was decreased after PKC? inhibitor treatment.Quantitative proteomic analysis identified a total of 3342 proteins.Among the DEHP treated MCF-7 cells,206 proteins were increased and 207 proteins were decreased;185 proteins were increased in DEHP treated 3?-HSD silencing cells.197 proteins were decreased;217 proteins were significantly increased and 223 proteins were significantly down-regulated in DEHP treated 3?-HSD overexpressing cells.Conclusion:1.DEHP can induce the up-regulation of m RNA and protein expression levels of 3?-HSD.There is a significant dose-effect relationship in the dose range of 0.1-0.8 mmol/L.There is a significant time-effect relationship at 48 h.DEHP may exert reproductive endocrine toxicity by affecting the expression level of 3?-HSD during the synthesis of steroid hormones.2.The 3?-HSD RNAi lentivirus vector was successfully constructed and the 3?-HSD silencing cell line was screened.The 3?-HSD gene overexpression vector was successfully constructed and the 3?-HSD overexpressing cell line was screened.3.DEHP can cause oxidative damage in MCF-7 cells,3?-HSD may play an antioxidant role in the process of oxidative damage of DEHP through affecting the activity of antioxidant enzymes and DNA damage repair process.4.DEHP can induce apoptosis in MCF-7 cells,3?-HSD may affect the expression of apoptotic genes and estrogen receptor genes,and play a role in promoting the apoptosis of DEHP.5.DEHP induced reproductive endocrine toxicity may be related to MAPK,NOS,and PKC signaling pathways,3?-HSD may regulate the expression of signaling pathway related genes,and DEHP may induce apoptosis through p38MAPK/i NOS signaling pathway;Reproductive toxicity may also be associated with cellular ribosome assembly and nucleosome assembly.