Label-free Quantitative Proteomics for Investigation of Signaling Pathways of GATA6 Regulating Trastuzumab Resistance in Gastric Cancer Cells

In this study, Crispr/Cas9 was employed to establish a GATA6 knock-out cell line (NCI N87R/GATA6). The signaling pathways regulated by GATA6 and related to trastuzumab resistance were investigated based on label-free quantitative proteomics combined with bioinformatics. The extracted proteins were alkylated, and digested using filter aided sample preparation (FASP), and the peptides were separated via high performance liquid chromatography (HPLC) and thereafter quantified by liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). Differentially expressed proteins were screened by fold change and student's t-test between NCI N87/R and NCI N87R/GATA6 cells. WebGestalt website was adopted for Gene ontology analysis, and GeneAnalytics database was utilized for pathway enrichment analysis. The results demonstrated that GATA6 knock-out enhanced the antiproliferative effect of trastuzumab on NCI N87/R cells and suppressed their invasion ability. A total of 5792 proteins were quantified by LC-MS/MS, among which 305 proteins were up-regulated in NCI N87R/GATA6 cells while 182 ones down-regulated. Pathway enrichment analysis revealed that mitochondrial transport, apoptosis, DNA damage, glucose metabolism, pyruvate metabolism and TCA cycle, and Wnt/β-catenin degradation pathways exhibited significant changes. Western blotting manifested that the expression of mitochondrial dyneins OPA1 and DNM1L, apoptosis protein caspase-9, TCA metabolic enzymes SUCLG2 and MDH1, and gl...
Source: Chinese Journal of Analytical Chemistry - Category: Chemistry Source Type: research