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.
目的： 探讨肿瘤坏死因子受体相关因子6(TRAF6)第331位泛素化位点突变对结直肠癌细胞生物学特征的影响及其相关机制。 方法： 将带有绿色荧光蛋白标签的TRAF6基因表达质粒的慢病毒(pCDH－3×FLAG－TRAF6)和突变质粒慢病毒(pCDH－3×FLAG －TRAF6－331mut)分别感染结直肠癌细胞SW480和HCT116，荧光显微镜观察细胞感染情况，Western blot检测TRAF6和TRAF6－331mut在细胞中的表达。采用细胞计数试剂盒8法和平板克隆实验检测TRAF6组和TRAF6－331mut组结直肠癌细胞的增殖能力，细胞划痕实验检测细胞的迁移能力，Transwell小室实验检测细胞的迁移和侵袭能力，免疫共沉淀方法检测TRAF6和TRAF6－331mut与泛素的赖氨酸结合位点K48和K63的泛素化作用，Western blot检测过表达TRAF6和TRAF6－331mut对核因子κB(NF－κB)和丝裂原活化蛋白激酶(MAPK)/激活蛋白1(AP－1)信号通路的影响。 结果： 荧光显微镜下观察到病毒成功感染结直肠癌细胞。Western blot检测显示，TRAF6和TRAF6－331mut在结直肠癌细胞中成功表达。CCK－8法检测结果显示，到第4天，TRAF6－331mut组HCT116和SW480细胞的A值分别为1.89±0.39和1.88±0.24，均低于TRAF6组(分别为2.09±0.12和2.17±0.45，P值分别为0.036和0.011)。平板克隆形成实验结果显示，TRAF6－331mut组HCT116和SW480细胞的克隆数分别为(120±14)个和(85±14)个，均低于TRAF6组[分别为(190±21)个和(125±13)个，P值分别为0.001和0.002]。细胞划痕实验结果显示，48 h后，TRAF6－331mut组HCT116和SW480细胞的伤口愈合距离百分比分别为(31±12)%和(33±14)%，均低于TRAF6组[分别为(43±13)%和(43±7)%，P值分别为0.005和0.009]。Transwell迁移实验结果显示，TRAF6－331mut组HCT116和SW480细胞的迁移数分别为(104±13)个和(107±12)个，均低于TRAF6组[分别为(172±19)个和(138±16)个，P值分别为<0.001和0.002]。Transwell侵袭实验结果显示，TRAF6－331mut组HCT116和SW480细胞的穿膜数分别为(103±17)个和(92±13)个，均低于TRAF6组[分别为(177±20)个和(127±18)个，P值分别为0.008和0.009]。免疫共沉淀法检测结果显示，在与K48共转染的293T细胞中，被TRAF6－331mut下拉的K48链泛素蛋白的表达量为0.57±0.19，低于野生型TRAF6下拉的K48链泛素蛋白的表达量(0.84±0.04，P＝0.014)；在与K63共转染的293T细胞中，被TRAF6－331mut下拉的K63链泛素蛋白的表达量为0.31±0.13，低于野生型TRAF6下拉的K63链泛素蛋白的表达量(0.89±0.08，P<0.001)。Western blot检测显示，TRAF6－331mut－HCT116细胞中NF－κB、p－NF－κB和p－AP－1蛋白表达水平分别为0.63±0.08、0.42±0.08和0.60±0.07，低于TRAF6－HCT116细胞(均为1.00±0.00，P值分别为0.002、<0.001和<0.001)，AP－1蛋白表达水平为0.89±0.06，与TRAF6－HCT116细胞(1.00±0.00)比较，差异无统计学意义(P＝0.051)；TRAF6－331mut－SW480细胞中NF－κB、p－NF－κB和p－AP－1蛋白表达水平分别为0.50±0.06、0.51±0.04和0.48±0.02，低于TRAF6－SW480细胞(均为1.00±0.00，均P<0.001)，AP－1蛋白表达水平为0.90±0.06，与TRAF6－ SW480细胞(1.00±0.00)比较，差异无统计学意义(P＝0.087)。 结论： TRAF6基因第331泛素化位点突变可能阻止了TRAF6与泛素赖氨酸位点K48和K63的结合，进而影响下游NF－κB和MAPKs/AP－1信号通路相关蛋白的表达，抑制结直肠癌细胞的增殖、迁移和侵袭能力。.
Keywords: Activator protein-1; Colorectal neoplasms; Mutant; Nuclear factor κ-B; Tumor necrosis factor receptor related factor 6; Ubiquitination.