关键词: 犬乳腺肿瘤细胞; 蛋白质组学; 差异表达蛋白; 代谢途径

Abstract:

Objective This study aimed to screen differentially expressed proteins between canine mammary tumor cells CHMM and CHMP， so as to provide scientific basis for the search of effective biomarkers for the diagnosis and treatment of canine mammary tumors. Method Using canine mammary tumor thoracic metastatic cells CHMM and primary cells CHMP as research objects， the morphologies， cloning ability， and migration ability of the two cell lines were compared through microscopic observation， cloning experiment， and Transwell experiment. Then， differentially expressed proteins in cells were screened using high-performance liquid chromatography-mass spectrometry analysis technology. GO function and KEGG pathway enrichment analysis on differentially expressed proteins was performed. Protein-protein interaction analysis was performed on differentially expressed proteins using STRING database. The proteomics results were validated through Western blotting experiment. Result CHMP cells exhibited a long spindle shape with single-cell growth， while CHMM cells grew in clusters with relatively small cells. CHMP cells had stronger migration ability， while CHMM cells had stronger cloning ability. A total of 2 049 proteins with differential expression were screened by sequencing （FoldChange>1.2 and P<0.05）， including 1 161 down regulated proteins and 888 upregulated proteins. GO functional enrichment analysis results showed that the differentially expressed proteins in cells involvement in biological processes mainly included cellular process， single-organism process， metabolic process，regulation of biological process， response to stimulus， etc. The cellular components included cell， cell part， organelle， organelle part， etc. Molecular functions mainly included catalytic activity， binding， and transporter activity. KEGG pathway enrichment analysis showed that the differentially expressed proteins in cells mainly participated in metabolic pathway signaling pathways. Differentially expressed proteins interaction analysis showed that CD44， MMP14 and DPYSL3 proteins had an interaction relationship. Western blotting showed that compared with CHMM cells， the expression level of GSDMD in CHMP cells significantly increased and the expression level of CD44 significantly decreased （P<0.05）， which was consistent with proteomic results，confirming the accuracy of sequencing. Conclusion Through proteomics screening， 1 161 downregulated proteins such as GSDMD and 888 upregulated proteins such as CD44 were identified in canine mammary tumor cells CHMM and CHMP， involving multiple biological processes such as metabolic pathways. The results of this study could provide scientific basis for the diagnosis and treatment of canine mammary tumors.

Key words: canine mammary gland tumor cells; proteomics; differentially expressed protein; metabolic pathways