The Role Of Aryl Hydrocarbon Receptor In MCF-7 Cell Migration Induced By Co-exposure To MEHP And TCDD

Objective:Our study was aimed at exploring the effects of co-exposure to MEHP and TCDD on cell proliferation, migration and invasion in breast cancer cell MCF-7, as well as on aryl hydrocarbon receptor signaling. Methods:1. MTS was used to analyze the cell proliferation after exposure to MEHP(0.1μM~100μM) and TCDD(1nM) during 6h, 12 h, 24 h and 48 h. Meanwhile, siRNA transfection was used to explore the role of AhR on the cell proliferation induced by TCDD and MEHP.2. Transwell assay was used to analyze the cell migration or invasion after exposure to MEHP(1μM~100μM) and TCDD(1nM) during 24 h and 48 h. Meanwhile, siRNA transfection was used to explore the role of AhR on the cell migration or invasion induced by TCDD and MEHP3. PCR-Array was used to screen genes related to tumor progression that might be induced by MEHP and TCDD.4. qRT-PCR and Western blotting were used to explore the expression of AhR, CYP1A1 and CYP1B1 induced by MEHP during 6h, 12 h, 24 h and 48 h, and induced by co-exposure of MEHP and TCDD during 24 h. P450-Glo? Assays were used to analyze the activity of CYP1A1 and CYP1B1 induced by MEHP and TCDD. In addition, Chromatin immunoprecipitation was used to investigate the AhR binding to DREs induced by MEHP and TCDD. Results:1. In MCF-7 cells, MEHP inhibited the cell proliferation in a U-curve model. After 6h of exposure in the presence of 10μM, 20μM, 40μM the MEHP inhibited cell proliferation(all with P < 0.001). After 48 h of exposure in the presence of 10μM, 20μM, 40μM the MEHP inhibited cell proliferation while other concentrations did not show the same effect(with P < 0.001, P = 0.002 and 0.003, respectively). The inhibition effects did not recover even after siRNA-AhR transfection. Those results indicated that the inhibition effects of MEHP on MCF-7 cells were AhR-independent.2. The cell proliferation was not affected after exposure to TCDD during 6h, 12 h, 24 h and 48h(P = 0.208, 0.875, 0.474 and 0.751, respectively). There was an interaction effect of co-exposure to MEHP and TCDD on cell proliferation after 6h and 48 h of exposure(P = 0.013, 0.011, respectively). After co-exposure to MEHP and TCDD, the cell proliferation was significantly lower than the controls. The inhibition effects did not recover even after siRNA-AhR transfection.3. MEHP promoted cell migration and invasion. The cell migration was significantly elevated after 24 h of exposure to MEHP 1μM, 10μM and 100μM(All with P ≤ 0.001) and afer 48 h of exposure to MEHP 10μM and 100μM(All with P < 0.001). Compared to MEHP 1μM, the cell invasion was also elevated afer 48 h of exposure to MEHP 10μM and 100μM(P = 0.028 and 0.003, respectively). The MEHP-induced the cell migration disappeared after siRNA-AhR transfection, while the invasion remains unaffected. Those results indicated that MEHP-induced cell migration was AhR-dependent, while MEHP-induced cell invasion was not. The results from qRT-PCR showed that MEHP could up-regulate the mRNA levels of MMP2, MMP9, Slug and Vimentin and down-regulate E-cadherin. The mRNA level of MMP2 was significantly reduced after siRNA-AhR transfection, indicating that the expression of MMP2 was AhR-dependent.4. The cell migration and invasion was also elevated after 24 h and 48 h exposure to TCDD 1 nM. There was an antagonistic interaction effect of co-exposure to MEHP and TCDD on cell migration. However, the antagonistic interaction of MEHP and TCDD on cell migration was disappeared after siRNA-AhR transfection. The mRNA levels of Slug and Vimentin were slightly lower in co-exposure to MEHP and TCDD than exposure to TCDD alone. In addition, the TCDD-induced expression of MMP2 and MMP9 were inhibited after siRNA-AhR transfection, indicating that TCDD-induced expression of MMP2 and MMP9 were AhR-dependent.5. Results from PCR-Array showed that MEHP could up-regulate mRNA levels of 23 genes and down-regulate mRNA levels of 8 genes. MEHP showed the same effects on 18 genes as TCDD, including 14 genes up-regulated and 4 genes down-regulated. Antagonistic interactions of co-exposure could be observed in 9 genes(MMP14, PAI1, PLAU, GPX1, DLL4, POU5F1, CXCR4, JUP and CYP1A1). Synergistic interactions could be seen in 5 genes(IL1B, CDH5, NFE2L2, MTOR and MAPK14).6. The mRNA level and transactivation of AhR were significantly up-regulated after 6h, 12 h, 24 h and 48 h exposure to MEHP(All with P ≤ 0.001). The mRNA levels of CYP1A1 and CYP1B1 were significantly up-regulated by MEHP in a dose- and time-dependent manner. MEHP-induced CYP1A1 and CYP1B1 were significantly reduced after siRNA-AhR transfection, indicating that MEHP induced CYP1A1 and CYP1B1 were AhR-dependent. Results from CHIP showed that MEHP could increase the binding of AhR to DRE in the promoter of CYP1A1 and enhancer of CYP1B1, indicating that MEHP could act as AhR agonist.7. There was an antagonistic interaction effect of co-exposure to MEHP and TCDD on the expression of AhR and CYP1A1(with P = 0.009 and 0.041, respectively). TCDD could inhibit MEHP-induced AhR expression, and MEHP could inhibit TCDD-induced CYP1A1 expression in an antagonistic manner, while MEHP increased TCDD-induced CYP1B1 expression in an additive manner. The antagonistic effect on CYP1A1 disappeared after siRNA-AhR transfection, while the additive effect on CYP1B1 remains unaffected. Results from CHIP showed that MEHP could inhibit TCDD-induced AhR binding to DRE. Those results indicated that the antagonistic interactions of MEHP and TCDD on the expression of CYP1A1 could be explained by that MEHP could inhibit TCDD-induced AhR binding to DRE. Conclusion:1. MEHP could act as AhR agonist and up-regulate expression and transactivation of AhR, which was one of the mechenisms that MEHP induced MCF-7 migration.2. MEHP could inhibit TCDD-induced AhR binding to DREs in an antagonistic way, through which MEHP inhibited TCDD-induced CYP1A1 mRNA levels.3. The antagonistic effects of co-exposure to MEHP and TCDD could be observed on the expression of MMP14, PAI1, PLAU, GPX1, DLL4, POU5F1, and CXCR4. The synergistic effects of co-exposure to MEHP and TCDD could be observed on the expression of IL1 B, CDH5, NFE2L2, MTOR and MAPK14.