The Role Of GPER In The Arsenic-induced Invasion And Migration Of Human Mammary Epithelial Cells And Its Underlying Mechanism

Objective: Arsenic is a metal-like species that is widely found in nature.Arsenic exposure in Drinking Water has become a major public health problem.Arsenic was identified as a Class I carcinogen by the International Cancer Center,but the carcinogenic mechanism of arsenic is still unclear.In recent years,a number of in vitro and in vivo experiments have proved that arsenic is also an environmental endocrine disruptor,and long-term low-dose arsenic exposure has estrogen like effect.Mammary gland is one of the main target organs of estrogen regulation.According to global tumor epidemic statistics,breast cancer has become the leading cause of cancer deaths in women around the world.The results of epidemiological investigations show that arsenic exposure is closely related to the occurrence of breast cancer.G-protein coupled estrogen receptor(GPER)is a membrane estrogen receptor that is widely present in multiple systems and tumor cells throughout the body.It mediates rapid non-genetic estrogen effects and regulates the growth of a variety of estrogen-related cancer cells.In this study,human normal mammary epithelial cells(MCF-10A)were exposed to inorganic arsenic at low levels for a long period of time.From the perspective of epithelial mesenchymal transition(EMT)and changes in cell invasion and migration capabilities to investigate the role of GPER in human normal breast epithelial cells malignant transformation induced by inorganic arsenic and its specific mechanism.Methods: 1.Long-term treatment of MCF-10 A cells with inorganic arsenic: Long-term treatment of MCF-10 A cells with 0.1 ?mol/L,0.5 ?mol/L,and 2 ?mol/L sodium arsenite(Na As O2,As),and the control group was unstained arsenic treated in the same period.Cell culture medium were changed every other day.When the cells grow to 80-90%,cell subculture was performed,Cells were frozen every two weeks.Cell samples were collected at 0,8,16,20,and 24 weeks after exposure.2.Detection of malignant transformation-related indicators: The scratch healing experiment was used to detect the change of cell migration ability at the 8,16 and 24 weeks after long-term exposure,and the modified Transwell experment was used to detect the change of cell invasion ability at the 24 weeks.3.EMT assessment: MCF-10 A cell protein samples were collected for 24 weeks of long-term arsenic exposure,use Western blot to detect E-cadherin and Vimentin protein expressions to evaluate cell EMT.4.Detection of GPER-EGFR-ERK1/2 signaling pathway related proteins: Cells were collected at different time points(0 weeks,8 weeks,16 weeks,20 weeks,24 weeks)for long-term exposure,and Western blot was used to detect GPER,EGFR,P-EGFR,ERK1/2,P-ERK1/2 protein expression levels.Results: 1.Wound-healing assay of MCF-10 A cells induced by long-term inorganic arsenic exposure: After 8 weeks of chronic arsenic exposure,there was no obvious difference in scratch healing abilities between different treatment groups compared with the control group.After 16 weeks of chronic arsenic exposure,the scratch healing speeds of the 0.1 ?mol/L,0.5 ?mol/L,and 2 ?mol/L treatment groups were faster than the control group,but there was no obvious difference.After 24 weeks of chronic arsenic exposure,the scratch healing speeds of the 0.1 ?mol/L,0.5 ?mol/L,and 2 ?mol/L treatment groups became faster,and the 0.5 ?mol/L and 2 ?mol/L treatment groups were obviously higher than the control group.2.Long-term inorganic arsenic exposure induced changes in cell invasion capacity of MCF-10 A cells: After 24 weeks of chronic arsenic exposure,compared with the control group the cell invasion ability of the 0.1 ?mol/L,0.5 ?mol/L and 2 ?mol/L treatment groups was significantly enhanced(P < 0.05).3.EMT in MCF-10 A cells induced by long-term inorganic arsenic exposure: After 24 weeks of chronic arsenic exposure,the expression levels of E-cadherin protein in the 0.1 ?mol/L,0.5 ?mol/L,and 2 ?mol/L treatment groups were significantly reduced compared with the control group(P < 0.01),the expression levels of Vimentin protein were significantly increased(P < 0.05).4.Activation of GPER-EGFR-ERK1/2 signaling pathway in MCF-10 A cells caused by long-term inorganic arsenic exposure: The expression of GPER protein in MCF-10 A cells was increased after 24 weeks of treatment with different concentrations of inorganic arsenic.Compared with the control group,the GPER protein expression level in the 0.1 ?mol/L treatment group was increased,but there was no statistically significant,the GPER expression level in the 0.5 ?mol/L and 2 ?mol/L treatment groups were significantly increased(P < 0.01).Compared with the control group,the expression level of P-EGFR in the 0.1 ?mol/L treatment group was increased,but there was no statistical difference,the expression levels of P-EGFR in the 0.5 ?mol/L and 2 ?mol/L treatment groups were significantly increased(P < 0.05).Compared with the control group,the expression level of P-ERK1/2 in the 0.1 ?mol/L treatment group was increased,but there was no statistical difference,the expression levels of P-ERK1/2 protein in the 0.5 ?mol/L and 2 ?mol/L treatment groups were significantly increased(P < 0.05).There was no statistical difference in the expression levels of EGFR and ERK1/2 protein in each of the above exposed groups.5.The relationship between exposure time and GPER-EGFR-ERK1/2 pathway activation: With the increase of exposure time,the expression level of GPER protein expression level was increased.Compared with the control group without inorganic arsenic treatment,the expression level of GPER protein was increased at 8 weeks of arsenic exposure,but there was no statistical difference.Compared with the control group without inorganic arsenic treatment,the expression levels of GPER protein were increased significantly at 16 weeks,20 weeks,and 24 weeks(P < 0.05).With the increase of exposure time,the expression of P-EGFR protein was increased.Compared with the control group without inorganic arsenic treatment,the expression level of P-EGFR protein was increased after 8 weeks of arsenic exposure,but there was no statistical difference.Compared with the control group without inorganic arsenic treatment,the expression level of P-EGFR protein of MCF-10 A cells at 16 weeks,20 weeks,and 24 weeks of exposure was significantly increased(P < 0.05).With the increase of exposure time,the expression level of P-ERK1/2 protein was increased.Compared with the control group without inorganic arsenic treatment,the expression level of P-ERK1/2 protein was increased at 8 weeks of arsenic exposure,but there was no statistical difference.Compared with the control group without inorganic arsenic treatment,the expression of P-ERK1/2 protein increased significantly at 16 weeks,20 weeks,and 24 weeks exposure(P < 0.01).There was no significant difference in EGFR and ERK1/2 protein expression levels at each time point.Conclusion: 1.Long-term exposure to inorganic arsenic at low levels induce the changes of invasion and migration ability of MCF-10 A cells.2.Long-term exposure to inorganic arsenic at low levels induce EMT in MCF-10 A cells.3.Long-term exposure to inorganic arsenic at low levels activate the GPER-EGFR-ERK1/2 pathway of MCF-10 A cells,which activation may be related to changes in cell invasion and migration abilities.