Background:Effective inhibition of pathological cardiac hypertrophy is critical for managing various cardiovascular diseases,especially in cold environments.The communication between cardiomyocytes and fibroblasts,med...Background:Effective inhibition of pathological cardiac hypertrophy is critical for managing various cardiovascular diseases,especially in cold environments.The communication between cardiomyocytes and fibroblasts,mediated by secreted proteins,plays a significant role in the development and progression of pathological cardiac hypertrophy.Serpin Family E Member 2(serpinE2),secreted by fibroblasts into the extracellular space,has been implicated in this process.However,whether serpinE2 can be internalized by cardiomyocytes and whether cold exposure influences this process remains unclear.Materials and methods:Mice were subjected to cold exposure(4°C,12 h/day for 8 weeks),and cardiac hypertrophy was induced by transverse aortic constriction(TAC).SerpinE2 expression was silenced by short interfering RNA(siRNA).Cardiac fibroblasts were stimulated with angiotensin II(Ang II)to induce serpinE2 secretion.Exogenous recombinant serpinE2,labeled with DyLight 488 or His-tag,was used to evaluate its internalization and functional role in cardiomyocytes.Internalization was inhibited by using antibodies against serpinE2,heparin,or endocytosis inhibitors(β-cyclodextrin,nystatin,dynasore,and chlorpromazine).Chromatin immunoprecipitation followed by quantitative polymerase chain reaction(PCR)was used to assess the binding of the transcription factor CDX1 to the serpinE2 promoter.Results:Cold exposure significantly increased serpinE2 mRNA and protein expression in mouse hearts.SerpinE2 levels were also upregulated in plasma and cardiac tissue following TAC.Knockdown of serpinE2 attenuated TAC-induced hypertrophy,restored left ventricular function,and reduced atrial natriuretic peptide,brain natriuretic peptide,andβ-myosin heavy chain fragment levels.Exogenous serpinE2 promoted cardiomyocyte hypertrophy,an effect that was reversed by serpinE2 knockdown.Co-culture with conditioned medium from Ang II-stimulated fibroblasts increased serpinE2 expression in cardiomyocytes.Exogenous serpinE2 was internalized via endocytosis,which was inhibited by antibodies,heparin,and endocytosis blockers.Internalized serpinE2 activated the protein kinase B(AKT)/β-catenin pathway in cardiomyocytes.CDX1 bound to the serpinE2 promoter and promoted its transcription in fibroblasts.CDX1 overexpression increased serpinE2 and collagen expression,while its suppression had the opposite effect.Administration of exogenous fibroblast growth factor 4(FGF4)or overexpression of FGF4 plasmid upregulated CDX1,serpinE2,and collagen expression in fibroblasts.Conclusions:SerpinE2 expression is responsive to cold stress and mediates intercellular communication between fibroblasts and cardiomyocytes.Fibroblast-secreted serpinE2 is internalized by cardiomyocytes via endocytosis,promoting hypertrophy through activation of the phosphatidylinositol 3-kinase(PI3K)-AKT/β-catenin pathway.The FGF4-CDX1 axis regulates serpinE2 expression and secretion in cardiac fibroblasts.展开更多
Objective： To investigated whether epigenetic mechanisms contribute to the variable expression of variable protease nexin1（PN-1） encoded by the SERPINE2 gene in different cell types. Methods： Working with 5 human ...Objective： To investigated whether epigenetic mechanisms contribute to the variable expression of variable protease nexin1（PN-1） encoded by the SERPINE2 gene in different cell types. Methods： Working with 5 human cell lines, we determined the CpG methylation status within two CpG islands in the SERPINE2 gene by bisulphate sequencing and the PN-1 mRNA level by Q-RT PCR. Results： A CpG island spanning the transcription initiation site showed little methylation in 3 of the cell lines and substantial methylation in 2 of the cell lines. A CpG island covering the translation starting site showed full methylation in all investigated cell lines. Methylation within the CpG island was not randomly distributed, but showed accumulation at specific sites. However, we were not able to distinguish any patterns which related the methylation frequency to the gene expression level. Inhibition of CpG methylation with 5-aza-2’-deoxycytidine led to a several fold increase in PN-1 mRNA levels, but based on the results on CpG methylation in the CpG island spanning the transcript, the effect is most likely indirect. Conclusion： We have carefully mapped the CpG methylation pattern in two CpG islands in the 5’ part of the SERPINE2 gene without finding any obvious inverse correlation between methylation frequency and expression level.展开更多
Background:Liver cancer is a malignancy with high morbidity and mortality rates.Serpin family E member 2(SERPINE2)has been reported to play a key role in the metastasis of many tumors.In this study,we aimed to investi...Background:Liver cancer is a malignancy with high morbidity and mortality rates.Serpin family E member 2(SERPINE2)has been reported to play a key role in the metastasis of many tumors.In this study,we aimed to investigate the potential mechanism of SERPINE2 in liver cancer metastasis.Methods:The Cancer Genome Atlas database(TCGA),including DNA methy-lation and transcriptome sequencing data,was utilized to identify the crucial oncogene associated with DNA methylation and cancer progression in liver can-cer.Data from the TCGA and RNA sequencing for 94 pairs of liver cancer tissues were used to explore the correlation between SERPINE2 expression and clin-ical parameters of patients.DNA methylation sequencing was used to detect the DNA methylation levels in liver cancer tissues and cells.RNA sequencing,cytokine assays,immunoprecipitation(IP)and mass spectrometry(MS)assays,protein stability assays,and ubiquitination assays were performed to explore the regulatory mechanism of SERPINE2 in liver cancer metastasis.Patient-derived xenografts and tumor organoid models were established to determine the role of SERPINE2 in the treatment of liver cancer using sorafenib.Results:Based on the public database screening,SERPINE2 was identified as a tumor promoter regulated by DNA methylation.SERPINE2 expression was significantly higher in liver cancer tissues and was associated with the dismal prognosis in patients with liver cancer.SERPINE2 promoted liver cancer metas-tasis by enhancing cell pseudopodia formation,cell adhesion,cancer-associated fibroblast activation,extracellular matrix remodeling,and angiogenesis.IP/MS assays confirmed that SERPINE2 activated epidermal growth factor receptor(EGFR)and its downstream signaling pathways by interacting with EGFR.Mechanistically,SERPINE2 inhibited EGFR ubiquitination and maintained its protein stability by competing with the E3 ubiquitin ligase,c-Cbl.Additionally,EGFR was activated in liver cancer cells after sorafenib treatment,and SER-PINE2 knockdown-induced EGFR downregulation significantly enhanced the therapeutic efficacy of sorafenib against liver cancer.Furthermore,we found that SERPINE2 knockdown also had a sensitizing effect on lenvatinib treatment.Conclusions:SERPINE2 promoted liver cancer metastasis by preventing EGFR degradation via c-Cbl-mediated ubiquitination,suggesting that inhibition of the SERPINE2-EGFR axis may be a potential target for liver cancer treatment.展开更多
Biomarkers play an important role in the detection at an early stage of pancreatic cancer. The aim of the present study was to optimize the conditions of antibody arrays for detecting Hippocalcin-like 1 （HPCAL1）, ph...Biomarkers play an important role in the detection at an early stage of pancreatic cancer. The aim of the present study was to optimize the conditions of antibody arrays for detecting Hippocalcin-like 1 （HPCAL1）, phosphatidylethanolamine binding protein 1 （PEBP1）, lectin galactoside-binding soluble 7 （LGALS7）, and serpin peptidase inhibitor clade E member 2 （SERPINE2） as biomarkers for pancreatic cancer detection in a single assay and to investigate antibodies’ specificity and cross-reactivity. Capture antibodies against HPCAL1, PEBP1, LGALS7 and SERPINE2 were printed on nitrocellulose coated glass slides. HPCAL1, PEBP1, LGALS7 and SERPINE2 proteins with different concentrations were incubated with the capture antibodies at different temperatures for different time periods. Biotinylated detection antibodies recognizing a different epitope on the captured proteins and a secondary detection molecule （Streptavidin-PE） were used to detect fluorescent signals. The arrays showed the strongest signals when the concentration of the capture antibodies was at 500 μg/mL in PBST0.05 （PBS with 0.05% Tween-20）, and the slides were incubated overnight at 4°C. The lowest protein concentration for detection was 2 ng/mL. Each antibody demonstrated high specificity to the corresponding antigen in detecting a mixture of 4 proteins without significant cross-reactivity. The fluorescence and biomarker concentration displayed a linear correlation. The antibody microarray system could be a useful tool for potential biomarker detection for pancreatic cancer.展开更多
基金supported by the National Natural Science Foundation of China(82370279,82170299,82330011,82003757).
文摘Background:Effective inhibition of pathological cardiac hypertrophy is critical for managing various cardiovascular diseases,especially in cold environments.The communication between cardiomyocytes and fibroblasts,mediated by secreted proteins,plays a significant role in the development and progression of pathological cardiac hypertrophy.Serpin Family E Member 2(serpinE2),secreted by fibroblasts into the extracellular space,has been implicated in this process.However,whether serpinE2 can be internalized by cardiomyocytes and whether cold exposure influences this process remains unclear.Materials and methods:Mice were subjected to cold exposure(4°C,12 h/day for 8 weeks),and cardiac hypertrophy was induced by transverse aortic constriction(TAC).SerpinE2 expression was silenced by short interfering RNA(siRNA).Cardiac fibroblasts were stimulated with angiotensin II(Ang II)to induce serpinE2 secretion.Exogenous recombinant serpinE2,labeled with DyLight 488 or His-tag,was used to evaluate its internalization and functional role in cardiomyocytes.Internalization was inhibited by using antibodies against serpinE2,heparin,or endocytosis inhibitors(β-cyclodextrin,nystatin,dynasore,and chlorpromazine).Chromatin immunoprecipitation followed by quantitative polymerase chain reaction(PCR)was used to assess the binding of the transcription factor CDX1 to the serpinE2 promoter.Results:Cold exposure significantly increased serpinE2 mRNA and protein expression in mouse hearts.SerpinE2 levels were also upregulated in plasma and cardiac tissue following TAC.Knockdown of serpinE2 attenuated TAC-induced hypertrophy,restored left ventricular function,and reduced atrial natriuretic peptide,brain natriuretic peptide,andβ-myosin heavy chain fragment levels.Exogenous serpinE2 promoted cardiomyocyte hypertrophy,an effect that was reversed by serpinE2 knockdown.Co-culture with conditioned medium from Ang II-stimulated fibroblasts increased serpinE2 expression in cardiomyocytes.Exogenous serpinE2 was internalized via endocytosis,which was inhibited by antibodies,heparin,and endocytosis blockers.Internalized serpinE2 activated the protein kinase B(AKT)/β-catenin pathway in cardiomyocytes.CDX1 bound to the serpinE2 promoter and promoted its transcription in fibroblasts.CDX1 overexpression increased serpinE2 and collagen expression,while its suppression had the opposite effect.Administration of exogenous fibroblast growth factor 4(FGF4)or overexpression of FGF4 plasmid upregulated CDX1,serpinE2,and collagen expression in fibroblasts.Conclusions:SerpinE2 expression is responsive to cold stress and mediates intercellular communication between fibroblasts and cardiomyocytes.Fibroblast-secreted serpinE2 is internalized by cardiomyocytes via endocytosis,promoting hypertrophy through activation of the phosphatidylinositol 3-kinase(PI3K)-AKT/β-catenin pathway.The FGF4-CDX1 axis regulates serpinE2 expression and secretion in cardiac fibroblasts.
基金supported by the Danish National Research Foundation (26-331-6)the Danish Cancer Society (DP 07043, DP 08001)Grosserer Alfred Nielsen and Hustrus Fond
文摘Objective： To investigated whether epigenetic mechanisms contribute to the variable expression of variable protease nexin1（PN-1） encoded by the SERPINE2 gene in different cell types. Methods： Working with 5 human cell lines, we determined the CpG methylation status within two CpG islands in the SERPINE2 gene by bisulphate sequencing and the PN-1 mRNA level by Q-RT PCR. Results： A CpG island spanning the transcription initiation site showed little methylation in 3 of the cell lines and substantial methylation in 2 of the cell lines. A CpG island covering the translation starting site showed full methylation in all investigated cell lines. Methylation within the CpG island was not randomly distributed, but showed accumulation at specific sites. However, we were not able to distinguish any patterns which related the methylation frequency to the gene expression level. Inhibition of CpG methylation with 5-aza-2’-deoxycytidine led to a several fold increase in PN-1 mRNA levels, but based on the results on CpG methylation in the CpG island spanning the transcript, the effect is most likely indirect. Conclusion： We have carefully mapped the CpG methylation pattern in two CpG islands in the 5’ part of the SERPINE2 gene without finding any obvious inverse correlation between methylation frequency and expression level.
基金This work was supported by grants from the National Nat-ural Science Foundation of China(82070652 and 81870434)Department of Science and Technology of Zhejiang Province(2020C04003)+2 种基金the Chinese Academy of Medi-cal Sciences(019-I2M-5-030)the Jinan Microecological Biomedicine Shandong Laboratory(JNL-2022007B)the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases(zz202302).
文摘Background:Liver cancer is a malignancy with high morbidity and mortality rates.Serpin family E member 2(SERPINE2)has been reported to play a key role in the metastasis of many tumors.In this study,we aimed to investigate the potential mechanism of SERPINE2 in liver cancer metastasis.Methods:The Cancer Genome Atlas database(TCGA),including DNA methy-lation and transcriptome sequencing data,was utilized to identify the crucial oncogene associated with DNA methylation and cancer progression in liver can-cer.Data from the TCGA and RNA sequencing for 94 pairs of liver cancer tissues were used to explore the correlation between SERPINE2 expression and clin-ical parameters of patients.DNA methylation sequencing was used to detect the DNA methylation levels in liver cancer tissues and cells.RNA sequencing,cytokine assays,immunoprecipitation(IP)and mass spectrometry(MS)assays,protein stability assays,and ubiquitination assays were performed to explore the regulatory mechanism of SERPINE2 in liver cancer metastasis.Patient-derived xenografts and tumor organoid models were established to determine the role of SERPINE2 in the treatment of liver cancer using sorafenib.Results:Based on the public database screening,SERPINE2 was identified as a tumor promoter regulated by DNA methylation.SERPINE2 expression was significantly higher in liver cancer tissues and was associated with the dismal prognosis in patients with liver cancer.SERPINE2 promoted liver cancer metas-tasis by enhancing cell pseudopodia formation,cell adhesion,cancer-associated fibroblast activation,extracellular matrix remodeling,and angiogenesis.IP/MS assays confirmed that SERPINE2 activated epidermal growth factor receptor(EGFR)and its downstream signaling pathways by interacting with EGFR.Mechanistically,SERPINE2 inhibited EGFR ubiquitination and maintained its protein stability by competing with the E3 ubiquitin ligase,c-Cbl.Additionally,EGFR was activated in liver cancer cells after sorafenib treatment,and SER-PINE2 knockdown-induced EGFR downregulation significantly enhanced the therapeutic efficacy of sorafenib against liver cancer.Furthermore,we found that SERPINE2 knockdown also had a sensitizing effect on lenvatinib treatment.Conclusions:SERPINE2 promoted liver cancer metastasis by preventing EGFR degradation via c-Cbl-mediated ubiquitination,suggesting that inhibition of the SERPINE2-EGFR axis may be a potential target for liver cancer treatment.
基金supported by the Lustgarten Foundation for Pancreatic Cancer Research (No. RFA-08-003)
文摘Biomarkers play an important role in the detection at an early stage of pancreatic cancer. The aim of the present study was to optimize the conditions of antibody arrays for detecting Hippocalcin-like 1 （HPCAL1）, phosphatidylethanolamine binding protein 1 （PEBP1）, lectin galactoside-binding soluble 7 （LGALS7）, and serpin peptidase inhibitor clade E member 2 （SERPINE2） as biomarkers for pancreatic cancer detection in a single assay and to investigate antibodies’ specificity and cross-reactivity. Capture antibodies against HPCAL1, PEBP1, LGALS7 and SERPINE2 were printed on nitrocellulose coated glass slides. HPCAL1, PEBP1, LGALS7 and SERPINE2 proteins with different concentrations were incubated with the capture antibodies at different temperatures for different time periods. Biotinylated detection antibodies recognizing a different epitope on the captured proteins and a secondary detection molecule （Streptavidin-PE） were used to detect fluorescent signals. The arrays showed the strongest signals when the concentration of the capture antibodies was at 500 μg/mL in PBST0.05 （PBS with 0.05% Tween-20）, and the slides were incubated overnight at 4°C. The lowest protein concentration for detection was 2 ng/mL. Each antibody demonstrated high specificity to the corresponding antigen in detecting a mixture of 4 proteins without significant cross-reactivity. The fluorescence and biomarker concentration displayed a linear correlation. The antibody microarray system could be a useful tool for potential biomarker detection for pancreatic cancer.
