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
Background:Increased Galectin 3-binding protein(LGALS3BP)serum levels have been used to assess hepatic fibrosis stages and the severity of hepatocellular carcinoma(HCC).Considering the crucial role of transforming gro...Background:Increased Galectin 3-binding protein(LGALS3BP)serum levels have been used to assess hepatic fibrosis stages and the severity of hepatocellular carcinoma(HCC).Considering the crucial role of transforming growth factor-β1(TGF-β1)in the emergence of these diseases,the present study tested the hypothesis that LGALS3BP regulates the TGF-β1 signaling pathway.Methods:The expression levels of LGALS3BP and TGFB1 were analyzed in patients with metabolic dysfunction-associated steatohepatitis(MASH)and HCC.Multiple omics techniques,such as RNA-sequencing,transposaseaccessible chromatin-sequencing assay,and liquid chromatography-tandem mass spectrometry proteomics,were used to identify the regulatory mechanisms for the LGALS3BP-TGF-β1 axis.The effects of altered TGF-β1 signaling by LGALS3BP were investigated in conditional LGALS3BP-knockin and LGALS3BP-knockout mice.Results:In patients with MASH and HCC,the levels of LGALS3BP and TGFB1 exhibited positive correlations.Stimulation of LGALS3BP by the inflammatory cytokine interferonαin HCC cells or ectopic overexpression of LGALS3BP in hepatocytes promoted the expression levels of TGFB1.Aggravated fibrosis was observed in the livers of hepatocyte-specific LGALS3BP-knockin mice,with increased TGFB1 levels.LGALS3BP directly bound to and assembled integrinαV,an integral mediator required for releasing active TGF-β1 from extracellular latent complex with the rearranged F-actin cytoskeleton.The released TGF-β1 activated JunB transcription factor,which in turn promoted the TGF-β1 positive feedback loop.LGALS3BP deletion in the hepatocytes downregulated TGF-β1 signaling and CCl4 induced fibrosis.Moreover,LGALS3BP depletion hindered hepatocarcinogenesis by limiting the availability of fibrogenic TGF-β1.Conclusion:LGALS3BP plays a crucial role in hepatic fibrosis and carcinogenesis by controlling the TGF-β1 signaling pathway,making it a promising therapeutic target in TGF-β1-related diseases.展开更多
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.
基金Bio&Medical Technology Development Program of the National Research Foundation,Grant/Award Numbers:NRF-2020M3A9G3080281,NRF-2020R1A5A2031185Korean Government。
文摘Background:Increased Galectin 3-binding protein(LGALS3BP)serum levels have been used to assess hepatic fibrosis stages and the severity of hepatocellular carcinoma(HCC).Considering the crucial role of transforming growth factor-β1(TGF-β1)in the emergence of these diseases,the present study tested the hypothesis that LGALS3BP regulates the TGF-β1 signaling pathway.Methods:The expression levels of LGALS3BP and TGFB1 were analyzed in patients with metabolic dysfunction-associated steatohepatitis(MASH)and HCC.Multiple omics techniques,such as RNA-sequencing,transposaseaccessible chromatin-sequencing assay,and liquid chromatography-tandem mass spectrometry proteomics,were used to identify the regulatory mechanisms for the LGALS3BP-TGF-β1 axis.The effects of altered TGF-β1 signaling by LGALS3BP were investigated in conditional LGALS3BP-knockin and LGALS3BP-knockout mice.Results:In patients with MASH and HCC,the levels of LGALS3BP and TGFB1 exhibited positive correlations.Stimulation of LGALS3BP by the inflammatory cytokine interferonαin HCC cells or ectopic overexpression of LGALS3BP in hepatocytes promoted the expression levels of TGFB1.Aggravated fibrosis was observed in the livers of hepatocyte-specific LGALS3BP-knockin mice,with increased TGFB1 levels.LGALS3BP directly bound to and assembled integrinαV,an integral mediator required for releasing active TGF-β1 from extracellular latent complex with the rearranged F-actin cytoskeleton.The released TGF-β1 activated JunB transcription factor,which in turn promoted the TGF-β1 positive feedback loop.LGALS3BP deletion in the hepatocytes downregulated TGF-β1 signaling and CCl4 induced fibrosis.Moreover,LGALS3BP depletion hindered hepatocarcinogenesis by limiting the availability of fibrogenic TGF-β1.Conclusion:LGALS3BP plays a crucial role in hepatic fibrosis and carcinogenesis by controlling the TGF-β1 signaling pathway,making it a promising therapeutic target in TGF-β1-related diseases.
