关键词: 皮肤溃疡, 糖尿病, 人类白细胞抗原F介导转录因子10, Wnt1诱导信号通路蛋白1, 类泛素化修饰, Wnt/β-联蛋白信号通路

Abstract: 【Abstract】 Objective To investigate the mechanisms of action of the ubiquitin-like protein human leukocyte antigen-F adjacent transcript 10 （FAT10） in the impaired healing of diabetic ulcers （DUs）, and to provide new targets for DU treatment. Methods?The human immortalized keratinocyte cell line HaCaT was treated with 400 μmol/L methylglyoxal （MGO） for 48 hours to establish a DU cell model （MGO group）, and untreated HaCaT cells served as the normal control （NC group）. FAT10-overexpressing cells （oe-FAT10 group） and their empty vector controls （oe-NC group） were constructed, along with FAT10-knockdown cells （si-FAT10 group） and their negative controls （si-NC group）. Co-immunoprecipitation combined with glutathione S-transferase （GST） pull-down assay was employed to verify the FAT10-mediated ubiquitin-like modification of Wnt1-inducible signaling pathway protein 1 （WISP1）. FAT10-overexpressing and FAT10-knockdown HaCaT cells （MGO + oe-FAT10 group, MGO + si-FAT10 group） and their corresponding control cells （MGO + oe-NC group, MGO + si-NC group） were treated with MGO and subjected to the following analyses: 5-bromo-2′-deoxyuridine （BrdU） labeling assay, wound healing assay, and flow cytometry were performed to evaluate the effects of FAT10 on the proliferation, migration, and apoptosis of DU cell models, respectively; enzyme-linked immunosorbent assay （ELISA） was conducted to assess the effects of FAT10 on inflammatory cytokine levels in DU cell models; real-time quantitative PCR （qPCR） and Western blot analysis were employed to determine the expression of FAT10, WISP1, and key proteins in the Wnt/β-catenin signaling pathway. Comparisons between two groups were performed using the two-independent-samples t test, and data from two-factor experiments involving MGO intervention and FAT10 overexpression were analyzed using factorial analysis of variance. Results?Compared with the NC group, the proliferation rate of HaCaT cells in the MGO group decreased to 54.10% ± 3.44%. Compared with the NC group, the MGO group exhibited a significantly decreased 24-hour migration rate （t = 104.29, P < 0.001）, but a significantly increased total apoptosis rate and elevated levels of tumor necrosis factor-α （TNF-α）, interleukin （IL）-1α, IL-1β, and IL-6 （all P < 0.05）, indicating successful establishment of the DU cell model. Co-immunoprecipitation combined with GST pull-down assay confirmed the interaction between FAT10 and WISP1. The mRNA and protein expression levels of WISP1 were significantly higher in the MGO + oe-FAT10 group than in the MGO + oe-NC group （t = 3.09, 3.72, P = 0.037, 0.021, respectively）. Factorial analysis of variance showed that after MGO treatment or FAT10 overexpression, the cell proliferation index and 24-hour migration rate significantly decreased （FMGO = 148.76, 118.63, both P < 0.001; FFAT10 = 52.55, 36.25, both P < 0.001, respectively）, the total apoptosis rate and levels of TNF-α, IL-1α, IL-1β, and IL-6 significantly increased （all P < 0.001）, the mRNA and protein expression levels of β-catenin, c-Myc, and cyclin D1 significantly decreased （all P < 0.001）, and the mRNA and protein expression levels of glycogen synthase kinase-3β （GSK-3β） significantly increased （all P < 0.001）; however, there was no significant interaction between MGO treatment and FAT10 overexpression （all P > 0.05）. Compared with the MGO + si-NC group, the MGO + si-FAT10 group showed a significantly higher cell proliferation index and 24-hour migration rate （t = 7.97, 5.43, P = 0.001, 0.006, respectively）, a significantly lower total apoptosis rate and significantly lower levels of TNF-α, IL-1α, IL-1β, and IL-6 （all P < 0.001）, significantly higher mRNA and protein expression levels of β-catenin, c-Myc, and cyclin D1 （all P < 0.05）, and significantly lower mRNA and protein expression levels of GSK-3β （t = 7.31, 9.50, P = 0.002, < 0.001, respectively）. Conclusion?The ubiquitin-like protein FAT10 may induce alterations in the Wnt/β-catenin signaling pathway through ubiquitin-like modification of WISP1, thereby mediating the impaired healing process of DUs, suggesting that FAT10 may be a potential therapeutic target for DUs.

Key words: Skin ulcer, Diabetes mellitus, FAT10, WISP1, Ubiquitin-like modification, Wnt/β-catenin signaling pathway