The molecular mechanism of GSDMB regulating the fate of intestinal epithelial cells
Objective To explore the molecular mechanism of Gasdermin B(GSDMB)regulating the fate of intestinal epithelial cells.Methods The human GSDMB plasmid was overexpressed into two human intestinal epithelial cell lines(NCM460 and HT-29 cells)and human colon-derived organoids.Western blotting was used to confirm the efficiency of electroporation.Cell counting kit(CCK8),cell apoptosis,and cell cycle by flow cytometry were performed to analyze the effect of GSDMB overexpression on cell function.Transcriptome sequencing was used to analyze the downstream effector molecules of GSDMB.T test was used to compare the data between the two groups.Results The overexpression of GSDMB protein in the two intestinal epithelial cell lines was successfully reconstructed.The absorbance value(A)of human intestinal epithelial cells overexpressing GSDMB protein[NCM460 cells:(1.17±0.01),HT-29 cells:(0.96±0.06)]was significantly lower than that of blank control cells[NCM460 cells:(1.67±0.12),HT-29 cells:(1.24±0.07)](t=7.24 and 5.46,P＜0.05).The number of apoptotic cells in the GSDMB overexpression group[NCM460 cells:(12.03±1.55),HT-29 cells:(29.30±4.48)]was significantly higher than that in the blank group[NCM460 cells:(4.96±1.74),HT-29 cells:(6.95±3.42)](t=5.26 and 6.97,P＜0.05).Cell cycle analysis showed that the ratio of cells at G0/G1 phase in the GSDMB overexpression group[NCM460 cells:(47.98±5.28)％,HT-29 cells:(38.04±3.45)％]was significantly lower than that in the control group[NCM460 cells:(59.54±3.90)％,HT-29 cells:(63.81±1.76)％](t=3.05 and 11.53,P＜0.05).Transcriptome sequencing results showed that the dual specificity phosphatase 4 and 6(DUSP4 and DUSP6)genes were significantly upregulated after GSDMB protein expression.Fluorescence quantitative PCR results confirmed that the relative expression levels of DUSP4(2.45±0.15)and DUSP6(4.34±0.22)in intestinal epithelial cells transfected with GSDMB were significantly higher than those in the control group(1.06±0.05 and 1.01±0.02)(t=15.08 and 26.52,P＜0.05).After GSDMB-expressing NCM460 cells were treated with the DUSP inhibitor BCI,the BCI treatment group had a significantly increased expression level of p-ERK compared to the control group[(1.14±0.17)vs.(0.58±0.12)](t=5.42,P=0.002);the A value(1.84±0.07)and G0/G1 phase ratio(59.83±2.17)％in the BCI treatment group were significantly higher than those in the non-treatment group[(1.52±0.10)and(52.10±2.23)％],and the number of apoptosis in the BCI treated group(7.60±0.56)was significantly lower than that in the untreated group(12.57±1.00)(t=4.71,4.31,7.52,P＜0.05).TUNEL staining in human colon organoids showed a significant increase in apoptotic cells,and the relative expression level of DUSP6 protein(0.85±0.09)was significantly higher than that of the control group(0.21±0.04),accompanied by a decrease in p-ERK levels[(0.83±0.18)vs.(0.19±0.06)],with statistical significance(t=11.95,P＜0.001;t=6.56,P＜0.001).Conclusion GSDMB may inhibit cell proliferation,induce cell cycle arrest,and promote apoptosis by upregulating dual specificity phosphatase DUSP6-mediated ERK phosphorylation,thus affecting the fate of intestinal epithelial cells.
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维普
