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• Research Paper Volume 12, Issue 9 pp 7679-7693

  Quantitative acetylome and phosphorylome analysis reveals Girdin affects pancreatic cancer progression through regulating Cortactin

  Relevance score: 8.053938

  Lihua Yang, Qiang Fu, Lin Miao, Quchen Ding, Xiangyu Li, Juan Wang, Guobin Jiang, Yun Wang

  Keywords: Girdin, acetylome, phosphorylome, pancreatic cancer, Cortactin

  Published in Aging on May 5, 2020

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  The actin-binding protein Girdin is involved in a variety of cellular processes, including pancreatic cancer. The objective of this study is to explore the role and the mechanism of Girdin in pancreatic cancer by quantitative acetylome and phosphorylome analysis. We firstly found that Girdin was overexpressed in pancreatic cancer tissue and increased expression of Girdin was associated with tumor size and stage of patients with pancreatic cancer. We established the shRNA knockdown of Girdin in PANC-1 and Aspc-1 cells, and we found that shGirdin inhibited proliferation, migration and invasion, and promoted apoptosis. Subsequently, we identified and quantified 5,338 phosphorylated sites in 2,263 proteins that changed in response to Girdin knockdown, and identified a similar set of Girdin-responsive acetylome data as well. Additional data revealed that down-regulation of Girdin affected Cortactin phosphorylation and acetylation, suggesting Cortactin as an important regulatory target of Girdin. Moreover, we found that overexpression of Cortactin could rescue the effect of shGirdin on proliferation, apoptosism, migration and invasion of pancreatic cancer cells. In general, our results provided new insights into the mechanisms of Girdin function including cell proliferation, migration and invasion, and offer biomarker candidates for clinical evaluation of Girdin.

  

  Expression of Girdin in pancreatic cancer tissues and cell lines. (A) The mRNA expression of Girdin in pancreatic cancer tissues and adjacent normal tissues was identified by RT-PCR analysis (n=41). (B, C) The protein expression of Girdin in pancreatic cancer tissues and adjacent normal tissues was examined by IHC analysis. (D) Girdin expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1 and PANC-1) was identified by western blot analysis and RT-PCR. (E) Girdin expression was knockdown by 5 shRNAs and Girdin expression was identified by western blot analysis. (F) AsPC-1 and PANC-1 cells were transfected with shGirdin plasmid, the transfection efficiency was examined by RT-PCR and western blot analysis. Data was shown as mean ± SD. ***P<0.001. The experiments were repeated 3 times.

  
  
  

  

  Girdin down-regulation regulates pancreatic cancer progression in vitro and in vivo. (A) Cell proliferation rates of both shCtrl and shGirdin cells were tested by CCK-8 assay. (B) APC/PI staining analysis to measure apoptosis levels of both shCtrl and shGirdin cells. Apoptosis levels were significantly higher in shGirdin cells. (C) Wound-healing assay was performed to examine the migration in PANC-1 and AsPC-1 cells with shCtrl or shGirdin transfection. (D) Transwell invasion assay was carried out in PANC-1 and AsPC-1 cells with shCtrl or shGirdin transfection. (E, F) Tumor growth curves were established by measuring tumor volume every 5 for 35 days after injection. Tumor weights isolated from nude mice in each treatment group were determined on day 28 after injection. Data was shown as mean ± SD. ***P<0.001. The experiments were repeated 3 times.

  
  
  

  

  Bioinformatic analysis of acetylome quantification. (A, B) The enrichment of up- and down-regulated proteins in GO including cellular component analysis, biological process analysis, and molecular function analysis. (C, D) The enrichment of up- and down-regulated proteins in KEGG pathways.

  
  
  

  

  Bioinformatic analysis of the quantified phosphorylome. (A, B) The enrichment of up- and down-regulated proteins in GO including cellular component analysis, biological process analysis, and molecular function analysis. (C) The enrichment of up- and down-regulated proteins in KEGG pathways.

  
  
  

  

  Crosstalk between quantitative phosphorylome and acetylome. (A) The protein-protein interaction network of phosphorylated and acetylated proteins. (B) The protein-protein interaction network clustered in the RNA spliceosome. (C) The protein-protein interaction network clustered in ribosome.

  
  
  

  

  Cortactin protein level is decreased when knocking down Girdin. (A, B) Cortactin phosphorylation and acetylation. Cortactin was phosphorylated at S47 (A) and acetylated at K161 (B). (C) Expression of Girdin and Cortactin transcripts following shRNA knockdown of Girdin in PANC-1 cells was assessed by qPCR. (D) Western blotting results showed Girdin and Cortactin protein expression levels in shCtrl cells and shGirdin cells, with GAPDH loading control. Data was shown as mean ± SD. ***P<0.001. The experiments were repeated 3 times.

  
  
  

  

  Cortactin over-expression reversed the effect of shGirdin on PANC-1 cells. PANC-1 cells were transfected with shNC, shGirdin+oeNC, and shGirdin+oeCortactin. (A) RT-PCR and western blot analysis were employed to detect the expressions of Girdin and Cortactin. (B–E) Cell proliferation, apoptosis, migration and invasion was identified by CCK8, APC/PI staining, wound healing, and transwell assays, respectively. Data was shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001. The experiments were repeated 3 times.