| Exposure to persistent organic pollutants(POPs),is closely related to human health,which is one of important risk factors for the occurrence and development of human cancers.Among POPs,endosulfan is one of organochlorine pesticides with the potential carcinogenicity.The epidemiologic studies found that there was a significant correlation between endosulfan and prostate cancer risk,but now there was no experimental evidence.Prostate cancer is one of the most common cancers in male,which belongs to epithelial malignancy with a short survival time and high mortality rate.Protein-tyrosine phosphatase4A3(PTP4A3)is considered to be a new inducing molecule of epithelial-mesenchymal transition(EMT)through various signaling pathways,contributing to cell migration and invasion as important mechanism of tumor metastasis.Our previous studies found that exposure to endosulfan elevated PTP4A3 expression,triggered EMT and promoted cell migration of prostate cancer cells.However,it remains unclear about the regulatory mechanism in up-regulation of PTP4A3 and signaling pathways of EMT when exposure to endosulfan.Therefore,the aim of this study is to explore the epigenetic mechanism in upregulation of PTP4A3 in DU145 and PC3 prostate cancer cells exposed by endosulfan,analyze signaling pathways mediated by PTP4A3 in endosulfan-induced EMT,and thus reveal the molecular mechanism in cell migration and invasion promoted by endosulfan in prostate cancer cells.In this study,there are three main contents.Firstly,we observed the effects of endosulfan on the proliferation,cell cycle,migration and invasion,then analyzed the signaling pathways of EMT induced by endosulfan,and focused on TGF-β and Notch signaling pathway.Secondly,we predicted the correlation and common genes between endosulfan exposure and human cancers.Then,we investigated whether PTP4A3 was involved in signaling pathways of EMT when endosulfan exposure using PTP4A3 inhibitor I and PTP4A3 plasmids including wild type and mutant.Finally,we predicted the regulatory relationship among lnc RNA,mi RNA and PTP4A3,and further analyzed the regulatory role of KCNQ1OT1/mi R-137-3p/PTP4A3 axis in EMT induced by endosulfan in prostate cancer cells.The results of this study were shown as follows:(1)The results from cell counting experiment showed that 5,10 and 20 μM endosulfan did not affect cell proliferation,but 40 μM endosulfan significantly inhibited cell proliferation.Therefore,5-20 μM endosulfan were chose in subsequent experiments.Using wound healing and Transwell assay we confirmed that endosulfan could significantly promote the ability of cell migration and invasion.q RT-PCR and Western blot results suggest that endosulfan exposure altered the expression of EMT biomarkers,including the downregulation of EMT epithelial marker(CDH1)and upregulation of mesenchymal markers(FN and Vimentin),transcription factors(SNAI2,ZEB2 and Twist1),key proteins(p-Smad2 and Smad2)in TGF-β signaling pathway and key proteins(Notch1 and Jagged1)in Notch signaling pathway.Above these alterations when exposure to endosulfan were interfered by TGF-β signaling pathway inhibitor(LY2109761)and Notch signaling pathway inhibitor(DAPT),respectively.These findings suggest that endosulfan activated TGF-β and Notch signaling pathways to induce EMT and thus promote cell migration and invasion.(2)Next Bio software was utilized to predict the correlation and the results showed that20 μM endosulfan exposure was associated with human breast cancer,prostate cancer,myeloid leukemia and uterine cancer.Importantly,PTP4A3 is found to be a common key gene among upregulated genes.Endosulfan exposure increased the expression of PTP4A3 at m RNA and protein levels in MCF-7,DU145,K562 and Si Ha cells,which were interfered by PTP4A3 inhibitor I.PTP4A3 inhibitor I suppressed the promotion of cell migration and invasion induced by endosulfan,expression changes of EMT biomarkers and key proteins in TGF-β signaling pathway,but it did not affect expression changes of the key proteins in Notch signaling pathway.After transfection with PTP4A3 plasmid into DU145 and PC3 cells,the expression of PTP4A3 protein was significantly increased.The abilities of cell migration and invasion were enhanced with obvious changes in EMT biomarkers and upregulation of key proteins in TGF-β signaling pathway after transfection with PTP4A3-WT plasmid but not the mutants.In addition,the expression of PTP4A3 was also up-regulated when exposed to dechlorane plus in concentration-independent manner.These findings suggest that PTP4A3 is a key molecule to induce EMT through TGF-β signaling pathway and thus promote the migration and invasion when exposure to endosulfan.(3)Bioinformatics analysis showed that there was complementary sequence in PTP4A33’-UTR and KCNQ1OT1 3’-UTR to the seed sequence of hsa-mi R-137-3p,so it was speculated that mi R-137-3p may target to regulate PTP4A3 and KCNQ1OT1.Luciferase reporter assay was used to confirm that PTP4A3 is the target gene of mi R-137-3p.Mi R-137-3p mimic significantly downregulated PTP4A3,while anti-mi R-137-3p upregulated PTP4A3,indicating that mi R-137-3p plays a negatively regulation role in PTP4A3 expression.Mi R-137-3p mimic caused the inhibition of migration and invasion in DU145 and PC3 cells,affected the changes in EMT biomarkers and the key proteins in TGF-β signaling pathway.Rescue experiment results showed that co-transfection of mi R-137-3p mimic and PTP4A3-WT plasmid reversed the results after transfection with mi R-137-3p mimic alone.The results of subcellular fractionation assay demonstrated the distribution of KCNQ1OT1 in cytoplasma.Evidence from RIP,RNA-RNA pull down and luciferase reporter assay showed that KCNQ1OT1 has an interaction with mi R-137-3p.The abilities of migration and invasion in DU145 and PC3 cells were inhibited,the expression changes of EMT biomarkers and key proteins in TGF-β signaling pathway were restrained after silencing of KCNQ1OT1,which were reversed by co-transfection with anti-mi R-137-3p or PTP4A3-WT plasmid.Overexpression of mi R-137-3p or silencing of KCNQ1OT1 significantly reduced the ability of cell migration and invasion,and suppressed the expression changes of EMT biomarkers and key proteins in TGF-β signaling pathway when exposure to endosulfan.These findings suggest that KCNQ1OT1/mi R-137-3p/PTP4A3 axis was involved in the regulation of endosulfan in the migration and invasion of prostate cancer cells.In summary,this study demonstrated that endosulfan triggered EMT in prostate cancer cells via TGF-β signaling pathway and Notch signaling pathway,and promoted cell migration and invasion.PTP4A3 is the key gene in EMT induced by endosulfan,contributing to the progression of many cancers.Endosulfan elevated PTP4A3 expression at post-transcriptional level through regulation of KCNQ1OT1-mi R-137-3p,and induced EMT via TGF-β signaling pathway to promote cell migration and invasion in prostate cancer cells.In the present study,endosulfan promoted cell migration and invasion with the induction of EMT through KCNQ1OT1/mi R-137-3p/PTP4A3 axis in prostate cancer cells,providing important value and experimental support for the study about the epigenetic and toxicological mechanism of typical POPs in the future. |
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