Study On The Effects Of TCDD/MEHP Exposure On Migration And Invasion In Breast Cancer Cell Line MCF-7 And Analysis Of The Mechanism

Objective:Breast cancer is the one of the most common malignant disease affecting women health. It is ranked the first in the incidence of cancer in women in our country. Metastasis is responsible for more than 90% breast cancer death. The initiation and progression of breast cancer is related to various risk factors. The factors related to environment and lifestyles are considered to contribute about 70%to 95% in overall risk factors. The purpose of this paper is to explore the typical environmental endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) and di-(2-ethylhexyl) phthalate(DEHP) metabolites mono-(2-ethyihexyl) phthalate(MEHP) alone and co-exposure on migration and invasion in breast cancer cell line MCF-7, and analyze the possible regulatory mechanism.Methods:1. The effects of TCDD/MEHP exposure on migration and invasion in MCF-7breast cancer cellsThe cells were treated with 1nM, 10 nM and 100 nM TCDD, and/or 100?M MEHP for 24 hours, and the effects of TCDD exposure on migration and invasion were tested by transwell migration and invasion assay.2. The effects of TCDD/MEHP exposure on migration and invasion genes m RNA. Total RNA was extracted from the cells treated with TCDD/MEHP at different concentrations for 24 hours. The expression of MMP2, MMP9, SLUG and VIM m RNA were assessed by real-time PCR.3. AhR pathway activation in TCDD-induced migration and invasion in MCF-7breast cancer cellsThe expression of Ah R and CYP1A1 m RNA was assessed by real-time PCR after the cells treated with TCDD at different concentrations for 24 hours.MCF-7 breast cancer cells were transfected with designed si RNA for Ah R and the non-targeting control si RNA for 48 hours. The transfection of Ah R m RNA expression was assessed by real-time PCR.To examine the role of Ah R pathway in mediating TCDD-induced migrationand invasion, MCF-7 breast cancer cells were transfected with designed si RNA for48 hours, and then treated with 100 n M TCDD for 24 hours. The changes of migration and invasion ability were tested by transwell migration and invasion assay.The changes of MMP2?MMP9, SLUG and Vim m RNA expression were assessed by real time PCR.Results:1. The effects of TCDD/MEHP exposure on migration and invasion in MCF-7breast cancer cellsTranswell migration and invasion assay showed that the ability of migration and invasion among treatment and control groups was statistically different(P<0.05).Compared with the control group, the ability of migration and invasion of 1 n M, 10 n M and 100 n M TCDD-treated groups were higher significantly(P<0.05). With the increase of TCDD exposure, the migration and invasion ability was established(P<0.05).Transwell migration and invasion assay showed that there were no significant differences of migration and invasion ability between 100?M MEHP-treated group and control group(P>0.05), but there was an increasing trend.Compared with the control group, the ability of migration and invasion of100?M MEHP and 100 n M TCDD co-treated group was higher significantly(P<0.05).When co-treated with 100?M MEHP and 100 n M TCDD, the ability of migration and invasion was different with the individual treatment group(P<0.05).2. The effects of TCDD/MEHP exposure on genes m RNA expression Real-time PCR revealed that 1 n M, 10 n M and 100 n M TCDD-treated groups up-regulated MMP2 and MMP9 m RNA relative expression levels compared to the control group significantly(P<0.05), and only 100 n M TCDD-treated significantly up-regulated SLUG and VIM m RNA relative expression levels(P<0.05).Real-time PCR revealed that 100?M MEHP exposure significantly up-regulated VIM m RNA relative expression levels significantly compared to the control group(P<0.05). There were no significant differences of MMP2, MMP9 and SLUG m RNA expression between 100?M MEHP-treated group and control group(P>0.05), but there was an increasing trend.When co-treated with 100?M MEHP and 100 n M TCDD, MMP2, MMP9 and VIM m RNA relative expression levels were higher significantly compared with the control group(P<0.05)3. Ah R pathway activated in TCDD-induced migration and invasion in MCF-7breast cancer cellsTo determine whether TCDD exposure activated the Ah R pathway in MCF-7breast cancer cells, real-time PCR revealed that there were no significant differences of Ah R m RNA relative expression levels in different concentrations(1, 10, 100 n M)TCDD-treated group compared to the control group(P>0.05). 1 n M, 10 n M and 100 n M TCDD-treated group up-regulated CYP1A1 m RNA relative expression levels significantly compared to control group(P<0.05).The transfection of si RNA for Ah R significantly reduced the Ah R relative m RNA expression levels 74% in comparison with the cells treated with the control si RNA(P<0.05), and reached the transfection efficiency. Transwell migration and invasion assay showed that MCF-7 cells transfected with Ah R and then exposed to100 n M TCDD decreased migration and invasion activity in comparison with the cells that transfected with control si RNA and then exposed to 100 n M TCDD.Real-time PCR revealed that MCF-7 cells transfected with Ah R and then exposed to100 n M TCDD decreased SLUG m RNA expression levels significantly(P<0.05),while there was a decreasing trend of MMP2, MMP9 and VIM m RNA expression levels(P>0.05).Conclusions:1. TCDD activated Ah R pathway and promoted migration and invasion of MCF-7 breast cancer cells by up-regulating MMP2, MMP9, SLUG and VIM m RNA relative expression levels.2. TCDD may induce epithelial-mesenchymal transition in MCF-7 breast cancer cells by up-regulating MMP2, MMP9, SLUG and VIM m RNA relative expression levels.3. MEHP may promote the migration and invasion of MCF-7 breast cancer cells.4. The effects of TCDD and MEHP co-exposure on migration and invasionwere different with individual exposure.