[Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells].

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Inhibition of TRAF6 improves hyperlipidemic acute pancreatitis by alleviating pyroptosis in vitro and in vivo rat models.

OBJECTIVE: Hypertriglyceridemia (HTG) is one of the common causes of acute pancreatitis (AP). Hyperlipidemic acute pancreatitis (HTG-AP) is associated with higher mortality owing to its tendency for greater severity and rapid progression. The purpose of this study was to explore the mechanism of involvement of tumor necrosis factor receptor-related factor 6 (TRAF6) in pyroptosis during HTG-AP. METHODS: The HTG environment was simulated with palmitic acid treatment in vitro and a high-fat diet in vivo. Cerulein was used to establish the HTG-AP model, followed by genetic and pharmacological inhibition of TRAF6. Pyroptosis activation, inflammatory reaction, and the interaction between TRAF6 and pyroptosis in HTG-AP were assessed. RESULTS: HTG was found to aggravate the development of pancreatitis, accompanied by increased pyroptosis and enhanced inflammatory response in HTG-AP models. Mechanistically, TRAF6 downregulation decreased the activation of pyroptosis in cerulein-induced HTG-AP. CONCLUSION: Collectively, inhibition of TRAF6 improved HTG-AP and the associated inflammation by alleviating pyroptosis.

Effect of TRAF6 in acute pancreatitis-induced intestinal barrier injury via TLR4/NF-κB signal pathway.

BACKGROUND: The aim of this study was to investigate the effect of tumor necrosis factor receptor-related factor 6 (TRAF6) in acute pancreatitis (AP)-induced intestinal barrier injury via the Toll-like receptor 4/nuclear factor kappa-B (TLR4/NF-κB) signal pathway. METHODS: Rat models of acute edematous pancreatitis (AEP) and acute necrotizing pancreatitis (ANP) were established by intraperitoneal injection of caerulein and retrograde infusion of sodium taurocholate solution into the biliopancreatic duct, respectively. Separate groups of model rats were pretreated with the TRAF6 inhibitor, MG-132. Rats were sacrificed at 12 h after the last injection for inducing AP. Histopathological changes, inflammatory response, intestinal barrier function, and protein expression levels were assessed by pathological score, ELISA, TUNEL, qRT-PCR, immunohistochemistry and western blotting. RESULTS: Rat models of AEP and ANP were successfully established as evidenced by the pathological changes in the pancreas and intestine. Pre-treatment with MG-132 significantly alleviated pancreatic and intestinal pathological scores, reduced serum levels of amylase, IL-1ß, and IL-6, and ameliorated apoptosis of mucosal cells. MG-132 reduced intestinal barrier injury, including serum levels of diamine oxidase and lipopolysaccharide, and intestinal expressions of ZO-1 and occludin. Moreover, it significantly suppressed the activation of the intestinal TLR4/NF-κB signaling pathway. CONCLUSIONS: TRAF6 inhibitor alleviated pancreatic and intestinal injury in AEP and ANP. This effect may be mediated through inhibition of the TLR4/NF-κB signaling pathway, which in turn regulates the inflammatory response and intestinal barrier injury.