High mobility group-box 3 overexpression is associated with poor prognosis of resected gastric adenocarcinoma

Abstract

Aim: To elucidate high mobility group-box 3 (HMGB3) protein expression in gastric adenocarcinoma, its potential prognostic relevance, and possible mechanism of action.

Methods: Ninety-two patients with gastric adenocarcinomas surgically removed entered the study. HMGB3 expression was determined by immunohistochemistry through a tissue microarray procedure. The clinicopathologic characteristics of all patients were recorded, and regular follow-up was made for all patients. The inter-relationship of HMGB3 expression with histological and clinical factors was analyzed using nonparametric tests. Survival analysis was carried out by Kaplan-Meier (log-rank) and multivariate Cox (Forward LR) analyses between the group with overexpression of HMGB3 and the group with low or no HMGB3 expression to determine the prognosis value of HMGB3 expression on overall survival. Further, HMGB3 expression was knocked down by small hairpin RNAs (shRNAs) in the human gastric cancer cell line BGC823 to observe its influence on cell biological characteristics. The MTT method was utilized to detect gastric cancer cell proliferation changes, and cell cycle distribution was analyzed by flow cytometry.

Results: Among 92 patients with gastric adenocarcinomas surgically removed in this study, high HMGB3 protein expression was detected in the gastric adenocarcinoma tissues vs peritumoral tissues (P < 0.001). Further correlation analysis with patients' clinical and histology variables revealed that HMGB3 overexpression was obviously associated with extensive wall penetration (P = 0.005), a positive nodal status (P = 0.004), and advanced tumor-node-metastasis (TNM) stage (P = 0.001). But there was no correlation between HMGB3 overexpression and the age and gender of the patient, tumor localization or histologic grade. Statistical Kaplan-Meier survival analysis disclosed significant differences in overall survival between the HMGB3 overexpression group and the HMGB3 no or low expression group (P = 0.006). The expected overall survival time was 31.00 ± 3.773 mo (95%CI = 23.605-38.395) for patients with HMGB3 overexpression and 49.074 ± 3.648 mo (95%CI = 41.925-57.311) for patients with HMGB3 no and low-level expression. Additionally, older age (P = 0.040), extensive wall penetration (P = 0.008), positive lymph node metastasis (P = 0.005), and advanced TNM tumor stage (P = 0.007) showed negative correlation with overall survival. Multivariate Cox regression analysis indicated that HMGB3 overexpression was an independent variable with respect to age, gender, histologic grade, extent of wall penetration, lymph nodal metastasis, and TNM stage for patients with resectable gastric adenocarcinomas with poor prognosis (hazard ratio = 2.791, 95%CI = 1.233-6.319, P = 0.019). In the gene function study, after HMGB3 was knocked down in the gastric cell line BGC823 by shRNA, the cell proliferation rate was reduced at 24 h, 48 h and 72 h. Compared to BGC823 shRNA-negative control (NC) cells, the cell proliferation rate in cells that had HMGB3 shRNA transfected was significantly decreased (P < 0.01). Finally, cell cycle analysis by FACS showed that BGC823 cells that had HMGB3 knocked down were blocked in G1/G0 phase. The percentage of cells in G1/G0 phase in BGC823 cells with shRNA-NC and with shRNA-HMGB3 was 46.84% ± 1.7%, and 73.03% ± 3.51% respectively (P = 0.001), whereas G2/M cells percentage decreased from 26.51% ± 0.83% to 17.8% ± 2.26%.

Conclusion: HMGB3 is likely to be a useful prognostic marker involved in gastric cancer disease onset and progression by regulating the cell cycle.

Keywords: Cell cycle; Cell proliferation; Gastric adenocarcinoma; High mobility group-box 3; Prognosis.

Figure 1

Expression of high mobility group-box 3 in gastric adenocarinoma tissue and peritumoral tissue. A: Peritumoral tissue, no staining; B: Peritumoral tissue, weak staining; C: Gastric adenocarinoma tissue, weak staining; D: Gastric adenocarcinoma tissue, highly positive staining.

Figure 2

Kaplan-Meier survival curves between high mobility group-box 3 high expression group and low expression group (P = 0.006, Mantel-cox). Group 1: High mobility group-box 3 (HMGB3) no or low expression; Group 2: HMGB3 overexpression.

Figure 3

Effects of small hairpin RNAs-high mobility group-box 3 on BGC823 cell proliferation by MTT assay. Data shown as mean ± SD. Experiment was performed in triplicate, P < 0.01 small hairpin RNAs-negative control (shRNAs-NC) vs shRNA-high mobility group-box 3 (HMGB3).

Figure 4

Flow cytometry analysis of cell cycle distribution in BGC823 cells after transfection of high mobility group-box 3 small hairpin RNA for 48 h. A: Small hairpin RNAs (shRNA)-negative control; B: shRNA-high mobility group-box 3 group.

Figure 5

Cell cycle distribution in BGC823 cells after transfection of high mobility group-box 3 small hairpin RNA for 48 h. Data shown as mean ± SD. Experiment was performed in triplicate, P = 0.001 between small hairpin RNAs-negative control (shRNAs-NC) and shRNA-high mobility group-box 3 (HMGB3).