Supplementary Material for: High-mobility group box 1 overexpression predicts a poor prognosis and promotes EMT in gastric cancer by activating TLR4/NF-κB signaling.

Objective The molecular mechanism of high-mobility group box 1(HMGB1) promoting the epithelial-mesenchymal transition (EMT) of gastric cancer(GC) has not been known well. This study aimed to explore the clinical effects of HMGB1 expression levels on the clinicopathological characteristics of patients with GC and to uncover the potential molecular mechanism by which promoting tumor progression. Methods The expression levels of HMGB1 in 125 pairs of patients with GC were detected by IHC and western blotting. Univariate and multivariate analyses were performed to evaluate the relationship between HMGB1 expression and clinical characteristics of patients with GC. Stable overexpression(Over-HMGB1) and knockdown(sh-HMGB1) GC cell lines(AGS and MKN-45) were used to determine the effects of HMGB1 on the activation of TLR4/NF-κB signaling. Differences were considered statistically significant at p<0.05 in two sides. Results HMGB1 is highly expressed in GC tissues and cell lines. High HMGB1 expression (HR=1.89, 95%CI:1.44-2.39, p=0.001) was an independent risk factor for OS in patients with GC. Downregulation of HMGB1 resulted in downregulation of TLR4 and NF-κB subunit (p-p65 and p-IκBα) expression, whereas the upregulated expression of HMGB1 led to increased expression of TLR4 and NF-κB subunits. Overexpression of HMGB1 promotes the upregulation of EMT-TF expression, which enhances the proliferation and migration abilities of GC cell lines. Conclusion HMGB1 is highly expressed in GC tissues and is associated with a poorer prognosis in patients with GC. HMGB1 activates the TLR4/NF-κB signaling pathway to promote EMT progression in GC cell lines. HMGB1 may be a critical molecule in prognosis prediction and a therapeutic target for patients with GC.

研究目的：高迁移率族蛋白B1（high-mobility group box 1, HMGB1）促进胃癌（gastric cancer, GC）上皮间质转化（epithelial-mesenchymal transition, EMT）的分子机制尚未完全阐明。本研究旨在探讨HMGB1表达水平对胃癌患者临床病理特征的影响，并揭示其促进肿瘤进展的潜在分子机制。
方法：采用免疫组化（immunohistochemistry, IHC）和蛋白质印迹法（western blotting）检测125例配对胃癌患者组织样本中HMGB1的表达水平。通过单因素及多因素分析评估HMGB1表达与胃癌患者临床特征的相关性。构建稳定过表达HMGB1（Over-HMGB1）及敲低HMGB1（sh-HMGB1）的胃癌细胞系（AGS和MKN-45），以明确HMGB1对TLR4/NF-κB信号通路活化的调控作用。采用双侧检验，以P<0.05为差异具有统计学意义。
结果：HMGB1在胃癌组织及细胞系中呈高表达。HMGB1高表达（风险比HR=1.89，95%置信区间CI:1.44~2.39，P=0.001）是胃癌患者总生存期（overall survival, OS）的独立危险因素。下调HMGB1表达可降低TLR4及NF-κB亚基（p-p65和p-IκBα）的表达水平，而上调HMGB1表达则可升高上述蛋白的表达量。过表达HMGB1可促进上皮间质转化相关转录因子（EMT-TF）的表达上调，进而增强胃癌细胞系的增殖与迁移能力。
结论：HMGB1在胃癌组织中高表达，且与胃癌患者不良预后密切相关。HMGB1可通过激活TLR4/NF-κB信号通路，促进胃癌细胞系的上皮间质转化进程。HMGB1有望成为胃癌患者预后预测的关键分子及治疗靶点。