X-Chromosome Linked Apoptosis Inhibitor-associated Protein Factor 1 Regulates Caspase 1-mediated Pyroptosis of Gastrointestinal Stromal Tumor Cells via Autophagy Pathway
Objective:To investigate the effect and mechanism of X-chromosome-linked apoptosis in-hibitor-associated protein factor 1(XAF1)on Caspase 1-mediated pyroptosis in human gastrointestinal stro-mal tumor(GIST)cells.Methods:Real-time fluorescent quantitative PCR and western blot were used to de-tect the expression levels of XAF1 in human normal gastric mucosal epithelial cell line(RGM-1)and GIST cell lines(GIST-T1,GIST-430,GIST-882).pcDNA3.1-XAF1 recombinant plasmid was transfected into GIST-882 cells by liposome-mediated transfection to construct GIST-882 cells with high XAF1 expression.GIST-882 cells were divided into control group,pcDNA3.1 group(transfected with pcDNA3.1 empty vector plasmid),pcDNA3.1-XAF1 group(transfected with pcDNA3.1-XAF1 recombinant plasmid),and pcD-NA3.1-XAF1+Rap group(transfected with pcDNA3.1-XAF1 recombinant plasmid and treated with autoph-agy activator rapamycin).Immunofluorescence staining was used to detect the expression of microtubule-asso-ciated protein 1 light chain 3(LC3)in each group of cells.Western blot was used to detect the LC3Ⅱ/LC3Ⅰ ratio and Beclin1 protein expression level in each group of cells.ELISA was used to detect the contents of IL-1β and IL-18 in the supernatant of each group of cells.Lactate dehydrogenase(LDH)release experiment was used to detect the LDH release rate of each group of cells.Western blot was used to detect the expression levels of Caspase-1 and its activated form cleaved-Caspase-1,NOD-like receptor pyrin domain-containing protein 3(NLRP3),and gasdermin D(GSDMD)protein in each group of cells.Results:Compared with RGM-1 cells,the relative expression levels of XAF1 mRNA and protein were significantly decreased in GIST-T1,GIST-430,and GIST-882 cells(P<0.05).After cell transfection,the relative expression levels of XAF1 mRNA and protein in GIST-882 cells of pcDNA3.1-XAF1 group were significantly higher than those of pcDNA3.1 group and control group(P<0.05).Compared with the control group,the LC3 fluorescence in-tensity in GIST-882 cells of pcDNA3.1-XAF1 group was significantly weakened,the LC3Ⅱ/LC3Ⅰ ratio and Beclin1 protein relative expression level were significantly decreased(P<0.05),the contents of IL-1β and IL-18 in the cell supernatant and LDH release rate were significantly increased(P<0.05),and the relative expression levels of Caspase-1,cleaved-Caspase-1,NLRP3,and GSDMD proteins were also significantly increased(P<0.05).Compared with the pcDNA3.1-XAF1 group,the LC3 fluorescence intensity in GIST-882 cells of the pcDNA3.1-XAF1+Rap group was significantly enhanced,the LC3Ⅱ/LC3Ⅰ ratio and Bec-lin1 protein relative expression level were significantly increased(P<0.05),and at the same time,the con-tents of IL-1β and IL-18 in the cell supernatant and LDH release rate were significantly decreased(P<0.05),and the relative expression levels of Caspase-1,cleaved-Caspase-1,NLRP3,and GSDMD proteins were also significantly decreased(P<0.05).Conclusion:XAF1 is downregulated in GIST cells.Overexpres-sion of XAF1 can promote Caspase 1-mediated pyroptosis of GIST cells by inhibiting autophagy and exert anti-tumor effects.
Gastrointestinal stromal tumorX-chromosome-linked inhibitor of apoptosis protein-related factor 1AutophagyCaspase 1Pyroptosis
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