| Objective:The incidence of thyroid carcinoma has been increasing in recent decades,and it has gradually become the most common solid malignant tumors in the endocrine system and in head and neck tumors.Bisphenol A?BPA?is a common environmental endocrine disruptors?EEDs?in daily life.In recent years,the impact of BPA on the development of thyroid carcinoma has gradually attracted people's attention.Papillary thyroid carcinoma?PTC?is the most common thyroid malignancy,and more than 50%of PTC patients have BRAFV600E gene mutations in their tumor tissues.The BRAFV600E600E mutation is closely related to the occurrence and development of PTC.In this study,we investigated whether BPA exposure could synergize with the BRAFV600E mutation to promote epithelial mesenchymal transition?EMT?of thyroid cells via ERK-Cox2pathway by clinical case studies and in vitro,which is of great significance for the biological behaviors including metastasis and invasion of thyroid carcinoma.We expect this study could provide new theoretical basis and scientific clues for reasonable treatment of PTC patients with BRAFV600E mutations.Methods:1.Clinical case study of thyroid carcinoma?1?After signing the informed consent form,blood samples and tumor tissues of 45 PTC patients and blood samples of50 healthy controls were collected.BRAFV600E mutation in PTC patients was detected by immunohistochemistry,and BPA content in serum was detected by ELISA.?2?The expression levels of E-cadherin,N-cadherin and MMP-9 mRNA were detected by qRT-PCR,and immunohistochemistry was used to examine the protein expression of EMT-related indicators E-cadherin,N-cadherin and MMP-9.2.In vitro cell assay verification?1?The BRAFV600E mutation transfected cell model Nthy-BRAFV600E was constructed using BRAF wild-type human thyroid follicular epithelial cells called Nthy-ori 3-1.?2?The transfected cells were treated with different concentrations of BPA,then the effects of BPA on cell migration,invasion and clone formation were analyzed.?3?Analyze the effects of different concentrations of BPA on the expression of BRAF,ERK,p-ERK,Cox2 and EMT-related indicators E-cadherin,N-cadherin and MMP-9 protein in transfected cells.Results:1.Compared with the BRAF wild-type patients,the mRNA and protein expression of N-cadherin and MMP-9 increased in tumors of PTC patients with BRAFV600E mutation,while the expression of E-cadherin decreased,which were related to BPA exposure.2.After different concentrations of BPA treatment,compared with Nthy-Vector cells?transfected empty vector?,the migration,invasion and clone formation abilities of Nthy-BRAFV600E cells were enhanced?P<0.05?.Compared with BPA control group,10-7 mol/L BPA enhanced cell migration,invasion and clone formation abilities?P<0.05?,while 3.33?10-4 mol/L BPA inhibited cell migration,invasion and clone formation abilities?P<0.05?.3.After different concentrations of BPA treatment,compared with Nthy-Vector cells,BRAF protein expression of Nthy-BRAFV600E cells increased significantly?P<0.05?,ERK phosphorylation level and Cox2,N-cadherin and MMP-9 protein expression levels of Nthy-BRAFV600E cells increased,while E-cadherin protein expression level of Nthy-BRAFV600E cells decreased?P<0.05?.Compared with BPA control group,10-7 mol/L BPA could promote ERK phosphorylation,meanwhile Cox2 and EMT-related molecule protein expression levels increased?P<0.05?,while3.33?10-4 mol/L BPA inhibited ERK phosphorylation,meanwhile Cox2 and EMT-related molecule protein expression levels decreased?P<0.05?.Conclusions:1.Compared with the BRAF wild-type group,PTC patients with BRAFV600E mutation are more prone to EMT process,and the increase of BPA exposure levels further promotes the occurrence of EMT process.2.Human exposure dose(10-7mol/L)of BPA can synergize with BRAFV600E mutation by activating ERK-Cox2signaling pathway to promote the occurrence of EMT in thyroid cells,thereby enhancing the ability of cell migration,invasion and clone formation. |
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