Molecular Mechanism Of Endosulfan-induced EMT Involving PTP4A3 In Prostate Cancer PC3 Cells

Endosulfan is a representative organochlorine pesticide,which belongs to typical persistent organic pollutants.The epidemiologic studies found that there was a significant correlation between endosulfan and prostate cancer risk,but now it is deficient in experimental evidence.It is known that protein tyrosine phosphatases(PTP4A3)is an important regulator for epithelial-mesenchymal transition(EMT),closely related to the development of cancer.Our previous studies had found that endosulfan(5-20 ?M)promoted the motility of prostate cancer PC3 cells,which was inhibited by PTP4A3 inhibitor.Therefore,the aim of this study is to explore the role of PTP4A3 on endosulfan-induced cell migration and invasion,so as to reveal the molecular mechanism of endosulfan-induced EMT in PC3 cells.In this study,whether Notch signaling pathway involved in cell migration induced by endosulfan in PC3 cells was investigated firstly.The results fromWound healing and Transwell assay confirmed that endosulfan could promote the ability of cell migration.qRT-PCR and Western blot results showed that endosulfan downregulated EMT epithelial marker CDH1,while mesenchymal markers(FN and VIM),transcription factors(SNAI2,ZEB2,Twist1)and the key genes(Cleaved-Notch1 and Jagged1)in Notch signaling pathway were upregulated.DAPT,a Notch signaling pathway inhibitor,interfered the above effects of endosulfan.Secondly,the effects of endosulfan on PTP4A3 gene expression,EMT phenotypes(cell migration and invasion,cytoskeleton,apoptosis,extracellular matrix,etc.),EMT biomarkers and signaling pathways in the presence of PTP4A3 inhibitor were studied.The results showed that endosulfan increased the mRNA/protein expression level of PTP4A3,promoted cell migration and invasion ability,downregulated cytoskeleton-related genes,and increased the expression levels of Smad2 and its phosphorylation in TGF-P signaling pathway.Notably,PTP4A3 inhibitor could inhibit the effects of endosulfan as mentioned above,affect TGF-? signaling pathway but did not affect Notch signaling pathway.Immunofluorescence results showed that endosulfan caused the nuclear location of ?-catenin that is key gene in Wnt/?-catenin signaling pathway,which was not influenced by PTP4A3 inhibitor,implying that PTP4A3 did not affect Wnt/p-catenin signaling pathway.Finally,the regulatory mechanism of endosulfan on PTP4A3 expression at post-transcription level was investigated.Predictive analysis found that PTP4A3 gene is potential target gene of hsa-miR-137.Exposure to endosulfan caused the downregulation of hsa-miR-137 expression.Anti-miR-137 increased the expression level of PTP4A3 while miR-137 mimic decreased PTP4A3 expression level.miR-137 could affect the expression alterations of PTP4A3,FN,Snail2,ZEB2,Smad2 and p-Smad2 in endosulfan-exposed PC3 cells,implying that miR-137 could be involved in the regulation of PTP4A3 gene expression caused by endosulfan,resulting in EMT through TGF-?/Smad2 signaling pathway.In summary,endosulfan caused EMT in PC3 cells through Notch signaling,Wnt/?-catenin signaling and TGF-?/Smad2 signaling pathways,resulting in the promotion of cell migration and invasion,and the expression alterations of EMT biomarkers.Endosulfan elevated PTP4A3 expression level via hsa-miR-137 and induced EMT through TGF-?/Smad2 signaling pathway.These findings provide the important evidence and reference value for revealing the relationship between endosulfan and prostate cancer.