Expression, Subcellular Location And Prognostic Relevance Of High Mobility Group Box1(HMGB1) In Hepatocellular Carcinoma

Part1Expression, Subcellular Location and Prognostic Relevance of High Mobility Group Box1(HMGB1) in Hepatocellular CarcinomaObjective High mobility group box1protein (HMGB1) plays dual roles as both a nuclear protein and a cytokine. The role of high mobility group box1in hepatocellular carcinoma (HCC) remains unknown. We examined the expression and subcellular location of HMGB1in normal liver tissues, HCC tissues and matched adjacent noncancerous tissues specimens to further understand the molecular pathogenesis of HCCMethods In this study, we examined HMGB1expression in HCC and corresponding adjacent noncancerous specimens obtained from155patients by immunohistochemistry, and in patients’serum by ELISA. Moreover, the association of HMGB1expression with clinicopathologic characteristics and survival was analyzed.Result In all normal liver tissues (18), HMGB1staining was positive and the HMGB1protein was mainly located in the nucleus of hepatocytes, rarely in the cytoplasm. Compared to the distribution of HMGB1in normal liver samples,76.6%(119/155) of adjacent noncancerous tissue samples showed diversiform translocation of HMGB1from nucleus to cytoplasm in hepatocytes. HMGB1stained positive in all tumor tissues, which was mainly localized in the nucleus. The level of nuclear HMGB1protein level was significantly higher compared to that of adjacent noncancerous tissues (p<0.001). In27.1%of these tumor specimens, cytoplasmic HMGB1staining was positive. Compared to healthy volunteers, HMGB1was significantly elevated in serum of patients with HCC. The level of nuclear HMGB1was significantly correlated with Edmondson-Steiner grade (P<0.001), TNM stage (P=0.001), HBV-DNA load (P=0.033), the severity of liver fibrosis (P=0.002), vessel invasion (P=0.027). Moreover, elevated cytoplasmic HMGB1in adjacent noncancerous tissues significantly correlated with high hepatitis B virus (HBV)-DNA load (p<0.001) and the presence of severe liver fibrosis (p<0.01). Multivariate analysis showed that high expression of cytoplasmic HMGB1in adjacent noncancerous tissue was an independent prognostic factor for disease-free survival (p=0.006, HR=2.022,95%CI:1.222-3.343) and overall survival (p=0.014, HR=2.102,95%CI:1.162-3.806).Conclusion Up-regulation of nuclear HMGB1is significantly related to poor prognosis but not an independent prognostic factor. However, HMGB1cytoplasmic translocation in adjacent noncancerous tissue of HCC is an independent prognostic factor for disease-free and overall survival in patients with HCC. After curative hepatectomy, future therapeutic strategies are likely to profit from the characterization of HMGB1expression and cellular distribution in surrounding noncancerous tissues. Part2Exogenous HMGB1promoted HCC cells migration and invasion in vitro.Objectives To explore the effect and the mechamism of exogenous HMGB1on HCC cells malignancy in vitro.Methods The effect of exogenous HMGB1on HCC cells proliferation and malignancy was assessed using cell counting kit-8, cell migration and invasion assay. To further confirm whether extracellular HMGB1was responsible for the effect in cell migration and invasion, HCC cells were treated with Glycyrrhizic acid (GA), the inhibitor of extracellular HMGB1. After treatment with indicated dose of HMGB1, the activation of ERK1/2was detected by western blot.Results Cell counting kit-8showed that exogenous HMGB1promoted HCC cells proliferation (p<0.05). Cell migration assay showed that HMGB1treatment promoted cell migration2.01-fold (±0.24) in Hep3B cells (p<0.01), and1.62-fold (±0.08) in Huh7cells (p<0.01). Similarly, exogenous HMGB1increased invasion of Huh7cells2.06-fold (±0.18)(p<0.01) through matrigel layer. We observed that blockade of exogenous HMGB1attenuated the increase in cell migration and invasion suggesting that the increase in cellular migration was dependent on HMGB1. Western-blot showed that HMGB1induced a rapid phosphorylation of ERK1/2in Hep3B and Huh7cells, but not in a dose-dependent or time-dependent manner. GA attenuated the activation of ERK1/2induced by HMGB1treatment in both Hep3B andHuh7cells.Conclusion Exogenous HMGB1promotes the HCC malignancy, probably in part through activation of ERK signaling.