Objectives:Progression to castration-resistant prostate cancer(CRPC)and metastasis are the greatest challenges to effective treatment.Anticancer strategies targeting the key kinases associated with the development of ...Objectives:Progression to castration-resistant prostate cancer(CRPC)and metastasis are the greatest challenges to effective treatment.Anticancer strategies targeting the key kinases associated with the development of CRPC may represent a breakthrough.The tyrosine kinase receptor Erythropoietin-producing hepatocellular(Eph)A2 receptor is highly expressed in CRPC cell lines and may be associated with tumor invasion and metastasis.However,the effects and exact mechanisms of EphA2 in CRPC are only partially understood.This study aimed to investigate the impact of EphA2 on CRPC cell behaviors and underlyingmolecular pathways.Methods:CRISPR/Cas9-mediated gene editing induced EphA2-disrupted in human-derived PC3 andDU145 cells.Single-guideRNAs(sgRNAs)targeting EphA2 were designed,and editing efficiency was validated.Optimal sgRNA sequences were selected to generate EphA2-knockdown(KD)and-overexpressing(OE)cell lines.Cell migration,proliferation,and apoptosis were assessed via functional assays.Transcriptomic analysis,quantitative PCR,and Western blotting were performed to identify downstream effectors.Bioinformatics analyses were used to correlate EphA2 and CDH1 expression with clinical parameters in prostate cancer patients.Results:Editing efficiency was found to vary among different sgRNAs targeting the EphA2 gene.EphA2-KD significantly inhibited CRPC cell migration but did not affect cell proliferation or apoptosis.Conversely,EphA2-OE significantly enhanced the migration of DU145 cells.Molecular analyses revealed that the expression of CDH1(an important marker of the epithelial-mesenchymal transition(EMT)in tumors)was significantly upregulated in PC3-EphA2-KD cells and downregulated in DU145-EphA2-OE cells,indicating that CDH1 is a downstream regulator of EphA2.Bioinformatic analysis revealed that higher EphA2 levels and lower CDH1 expression were both associated with an advanced tumor T stage,higher Gleason scores,and lymph nodemetastases in prostate cancer patients.More importantly,EphA2 was found to be an important predictor of lymph nodemetastasis,in addition to the Gleason score.Adding EphA2 to the Gleason score could significantly improve the detection of lymph node metastasis.Conclusion:CRISPR/Cas9-mediated EphA2-KD significantly suppressed the migration of CRPC cells through the inhibition of the EphA2-CDH1 axis.Strategies targeting the EphA2 genemay be promising for the treatment of CRPC.展开更多
AIM:To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity.METHODS:A four-generation Chinese family affected with autosomal dominant congenital cataract ...AIM:To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity.METHODS:A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited.Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals.Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery.The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software.Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2(EPHA2).Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins.The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction(qRT-PCR)and Western blotting assay,respectively.The cell migration was analyzed by wound healing assay.Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo.RESULTS:A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif(SAM)domain of EPHA2.Functional studies demonstrated the variant’s impact:reduced EPHA2 protein expression,altered subcellular localization,and disrupted interactions with other lens membrane proteins.This mutant notably enhanced human lens epithelial cell migration,and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast(DIC)optics.CONCLUSION:Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.展开更多
基金funded by the National Natural Science Foundation of China(Grant No.81801754)the Suzhou Science and Technology Bureau Development Plan(Grant No.SYS2020147)the 26th Batch of Extra-Curricular Academic Research Fund Project of Soochow University(Grant No.KY2024235B).
文摘Objectives:Progression to castration-resistant prostate cancer(CRPC)and metastasis are the greatest challenges to effective treatment.Anticancer strategies targeting the key kinases associated with the development of CRPC may represent a breakthrough.The tyrosine kinase receptor Erythropoietin-producing hepatocellular(Eph)A2 receptor is highly expressed in CRPC cell lines and may be associated with tumor invasion and metastasis.However,the effects and exact mechanisms of EphA2 in CRPC are only partially understood.This study aimed to investigate the impact of EphA2 on CRPC cell behaviors and underlyingmolecular pathways.Methods:CRISPR/Cas9-mediated gene editing induced EphA2-disrupted in human-derived PC3 andDU145 cells.Single-guideRNAs(sgRNAs)targeting EphA2 were designed,and editing efficiency was validated.Optimal sgRNA sequences were selected to generate EphA2-knockdown(KD)and-overexpressing(OE)cell lines.Cell migration,proliferation,and apoptosis were assessed via functional assays.Transcriptomic analysis,quantitative PCR,and Western blotting were performed to identify downstream effectors.Bioinformatics analyses were used to correlate EphA2 and CDH1 expression with clinical parameters in prostate cancer patients.Results:Editing efficiency was found to vary among different sgRNAs targeting the EphA2 gene.EphA2-KD significantly inhibited CRPC cell migration but did not affect cell proliferation or apoptosis.Conversely,EphA2-OE significantly enhanced the migration of DU145 cells.Molecular analyses revealed that the expression of CDH1(an important marker of the epithelial-mesenchymal transition(EMT)in tumors)was significantly upregulated in PC3-EphA2-KD cells and downregulated in DU145-EphA2-OE cells,indicating that CDH1 is a downstream regulator of EphA2.Bioinformatic analysis revealed that higher EphA2 levels and lower CDH1 expression were both associated with an advanced tumor T stage,higher Gleason scores,and lymph nodemetastases in prostate cancer patients.More importantly,EphA2 was found to be an important predictor of lymph nodemetastasis,in addition to the Gleason score.Adding EphA2 to the Gleason score could significantly improve the detection of lymph node metastasis.Conclusion:CRISPR/Cas9-mediated EphA2-KD significantly suppressed the migration of CRPC cells through the inhibition of the EphA2-CDH1 axis.Strategies targeting the EphA2 genemay be promising for the treatment of CRPC.
基金Supported by the Natural Science Foundation of Fujian Province(No.2021J01229)National Key Research and Development Program of China(No.2016YFC1000307).
文摘AIM:To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity.METHODS:A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited.Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals.Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery.The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software.Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2(EPHA2).Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins.The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction(qRT-PCR)and Western blotting assay,respectively.The cell migration was analyzed by wound healing assay.Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo.RESULTS:A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif(SAM)domain of EPHA2.Functional studies demonstrated the variant’s impact:reduced EPHA2 protein expression,altered subcellular localization,and disrupted interactions with other lens membrane proteins.This mutant notably enhanced human lens epithelial cell migration,and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast(DIC)optics.CONCLUSION:Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.
