| Objective:Inorganic arsenic exposure is a global public health problem,which can affect multiple organs of the body and cause serious damage.Epidemiological evidence suggests that the occurrence of breast cancer is related to arsenic exposure.Our previous experiment found that long-term low-dose arsenic exposure could induce malignant transformation of human mammary epithelial cells.Invasion and migration are the main characteristics of malignant transformed cells,and focal adhesion formation and cytoskeletal rearrangement play an important role in this process.Focal adhesion kinase（FAK）is considered to be the main kinase that regulates the dynamic changes of focal adhesion,which can cause the rearrangement of the fibrin skeleton and promote the formation and extension of filopodia.G protein-coupled estrogen receptor（GPER）,as an estrogen membrane receptor,is involved in the occurrence and development of breast cancer,but its role and mechanism in arsenic-induced malignant transformation of mammary epithelial cells need to be further confirmed.In this study,we used the inorganic arsenic-induced malignant transformed human mammary epithelial cells established by our group combined with specific protein inhibitors and agonists to explore the role and possible mechanism of GPER in arsenic-induced malignant transformation from the perspective of cytoskeletal rearrangement and focal adhesion changes.The aiming of this study was to provide a new theoretical basis for the relationship between arsenic and breast cancer.Methods:1.Cell culture and arsenic treatment:Normal human mammary epithelial cells（MCF-10A）were treated with 0.5μM sodium arsenite（Na As O2）,and the control group（Cont）was the arsenic-free cells treated in the same period.New medium was changed every other day,and the cells were passaged until the degree of fusion reached 80-90%.Cell samples were collected at 0,8,16,20 and 24 weeks of exposure.2.Cell groups and treatment:in the GEPR specific inhibitor（G15）intervention experiment,the cells were divided into 4 groups:Cont,0.5μM,Cont+G15,0.5μM+G15;In the intervention experiment with FAK specific agonist（Zn27）,the cells were divided into 5 groups:Cont,0.5μM,0.5μM+G15,0.5μM+G15+Zn27,Cont+Zn27.The intervention dose of G15 was 4μM,and the treatment duration was48 hours.The intervention dose of Zn27 was 20 n M and the treatment duration was 24h.3.Identification of cell malignant phenotype:cell migration ability was evaluated by scratch test,and cell invasion ability was evaluated by RTCA Transwell assay.4.Evaluation of cytoskeleton and focal adhesion:F-actin and Vinculin were observed by immunofluorescence and laser confocal microscopy,and the number and length of pseudopodia as well as the number of focal adhesion were counted respectively.5.FAK phosphorylation related protein:The protein expressions of p-FAK（Tyr397）,p-FAK（Tyr576/577）,p-FAK（Tyr925）,FAK,p-Src（Tyr416）and Src were detected by Western Bolt,and the protein bands were quantified by Image J software.6.Statistical analysis:All data in this study were analyzed by Graph Pad Prism 8.0.2software.Results:1.The expression of GPER protein was significantly up-regulated in the process of malignant transformation of cells induced by arsenic:compared with the cells exposed to 0.5μM arsenic for 0 week,the expression of GPER protein in the cells exposed to arsenic for 16 weeks,20 weeks and 24 weeks was significantly increased（P<0.05）.2.G15 significantly inhibited the malignant phenotype,cytoskeletal rearrangement and focal adhesion formation of arsenic-transformed cells:Compared with Cont cells,the scratch healing ability and cell invasion ability of cells exposed to 0.5μM arsenic for24 weeks were significantly enhanced,the average number of filopodia was significantly increased,the average length of filopodia was significantly increased,and the number of cell adhesion spot was significantly increased（P<0.05）;Compared with0.5μM arsenic exposed cells,all above indicators in 0.5μM+G15 intervention group were significantly inhibited,and these differences were statistically significant（P<0.05）.3.The expression of FAK phosphorylation-related proteins was significantly up-regulated during arsenic-induced malignant transformation of cells:compared with Cont cells,the protein expressions of p-FAK（Tyr397）/FAK,p-FAK（Tyr576/577）/FAK,p-FAK（Tyr925）/FAK and p-Src（Tyr416）/Src in 0.5μM arsenic-exposed cells were significantly increased（P<0.05）.The expressions of p-FAK（Tyr397）/FAK,p-FAK（Tyr576/577）/FAK in cells exposed to arsenic for 16,20 and 24 weeks were significantly higher than those in cells exposed to 0.5μM arsenic for 0 week（P<0.05）.The protein expressions of p-FAK（Tyr925）/FAK and p-Src（Tyr416）/Src were significantly increased（P<0.05）.4.G15 intervention significantly inhibited FAK phosphorylation-related proteins:compared with the cells exposed to 0.5μM arsenic for 24 weeks,the expressions of p-FAK（Tyr397）/FAK,p-FAK（Tyr576/577）/FAK,p-FAK（Tyr397）/FAK,p-FAK（Tyr576/577）/FAK in cells treated with 0.5μM+G15 were significantly increased.The expressions of p-FAK（Tyr925）/FAK and p-Src（Tyr416）/Src were significantly inhibited（P<0.05）.5.Zn27 intervention reversed the inhibitory effects of G15 on the malignant phenotype,cytoskeletal rearrangement and focal adhesion formation of arsenic-transformed cells:compared with the 0.5μM+G15 group,cells in the 0.5μM+G15+Zn27 group showed significantly enhanced scratch healing ability and invasion ability,significantly increased the average number of filopodia at the cell edge,significantly increased the average length of filopodia,and significantly increased the number of focal adhesion（P<0.05）.Conclusion:1.GPER and FAK were gradually activated during the malignant transformation of human mammary epithelial cells induced by long-term arsenic exposure,and the protein change tendency of GPER and FAK were consistent.2.In arsenic-transformed cells,GPER mediates cytoskeletal rearrangement and focal adhesion changes by regulating FAK activation,thereby affecting the migration and invasion ability of arsenic-transformed cells. |
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