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. 2014 May;390(1-2):271-80.
doi: 10.1007/s11010-014-1978-6. Epub 2014 Feb 9.

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines

Ruo-Chan Chen  1 , Pan-Pan Yi, Rong-Rong Zhou, Mei-Fang Xiao, Ze-Bing Huang, Dao-Lin Tang, Yan Huang, Xue-Gong Fan
Affiliations

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines

Ruo-Chan Chen et al. Mol Cell Biochem. 2014 May.

Abstract

High mobility group protein box1 (HMGB1) and its receptor-receptor for advanced glycation end products (RAGE) are pivotal factors in the development and progression of many types of tumor, but the role of HMGB1-RAGE axis in hepatocellular carcinoma (HCC) especially its effects on metastasis and recurrence remains obscure. Here, we report the role of HMGB1-RAGE axis in the biological behaviors of HCC cell lines and the underlying molecular mechanism. We show that the expressions of HMGB1, RAGE, and extracellular HMGB1 increase consistently according to cell metastasis potentials, while the concentration of soluble form of RAGE (sRAGE) is inversely related to metastasis potential of HCC cells. Furthermore, our data show that rhHMGB1 promotes cellular proliferation, migration, and invasion, and increases the level of nuclear factor kappa B (NF-κB), while administrations of HMGB1-siRNA, RAGE-siRNA, anti-HMGB1 neutralizing antibody, anti-RAGE neutralizing antibody, and sRAGE inhibit cellular proliferation, migration, and invasion. Moreover, we also demonstrate that the expression of NF-кB is inhibited by knockdown of HMGB1 or RAGE. Collectively, these data demonstrate that HMGB1 activates RAGE signaling pathways and induces NF-кB activation to promote cellular proliferation, invasion, and metastasis, in HCC cell lines. Taken together, HMGB1-RAGE axis may become a potential target in HCC therapy.

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Figures

Fig. 1
Fig. 1
HMGB1 and RAGE were expressed highly in HCCLM3 cells. a Relative expression of HMGB1 or RAGE mRNA normalized to β-actin was measured by qRT-PCR in Hep3B, MHCC97L, and HCCLM3 cells. b Expression of HMGB1 or RAGE protein in the three HCC cell lines was examined by Western blot. GAPDH was used as an internal control for quantity analysis. (**P < 0.01, *P < 0.05)
Fig. 2
Fig. 2
Extracellular HMGB1, and sRAGE level in the supernatants of HCC cells. The release level of extracellular HMGB1 (a) and sRAGE (b) was measured by ELISA in the supernatants of Hep3B, MHCC97L, and HCCLM3 cells. (*P < 0.05, **P < 0.01)
Fig. 3
Fig. 3
Effects of HMGB1 or RAGE antibodies, sRAGE on the survival rate of HCCLM3 cells. a Cell viability of HCCLM3 cells was analyzed by MTT after interacting with anti-RAGE neutralizing antibody, or human recombinant sRAGE at various concentrations (50, 100, 150, and 200 ng/ml), respectively for 24 h. RAGE antibody or sRAGE reduced the cell viability of HCCLM3 cells dose dependently, and both achieved the greatest growth inhibition at 150 ng/ml. b HCCLM3 cells were treated with anti-HMGB1 neutralizing antibody (20 ng/ml), anti-RAGE neutralizing antibody (150 ng/ml), and sRAGE (150 ng/ml), respectively for 24–72 h. Living rate of HCCLM3 cells was decreased by HMGB1 or RAGE antibody and sRAGE after incubating for 24 h and reached the greatest surviving inhibition after 72 h. c HCCLM3 cells were incubated with rhHMGB1 (50 ng/ml) for 48 h, then MTT assay showed that HMGB1 increased the living rate of HCCLM3 cells. (*P < 0.05, **P < 0.01)
Fig. 4
Fig. 4
HMGB1 siRNA and RAGE siRNA decreased the cell viability of HCCLM3 cells. a Knockdown of HMGB1 or RAGE in HCCLM3 cells by specific gene-targeted HMGB1-siRNA and RAGE-siRNA was confirmed by qRT-PCR. Add a descriptive label of the figure here. b After incubating with HMGB1-siRNA (75 ng/ml), RAGE-siRNA (75 ng/ml), and rhHMGB1 (50 ng/ml) for 48 h, the results of MTT assay showed that knockdown of HMGB1 or RAGE decreased the cell viability of HCCLM3 cells (*P < 0.05, **P < 0.01). NC negative control with a nonsense siRNA sequence
Fig. 5
Fig. 5
HMGB1 siRNA and RAGE siRNA attenuated invasion and mobility of HCCLM3 cells in vitro. HCCLM3 cells were seeded into the upper chamber of the transwell, treated with HMGB1-siRNA, RAGE-siRNA, anti-RAGE antibody or sRAGE, and rhHMGB1, and allowed to invade matrigel for 24 h. a The invasive cells migrating through the basal membrane to its lower surface were stained with crystal violet, then were photographed (20 × 10). b The number of invasive cells was also quantified by dissolving the purple crystals on the membranes in 500 μl 10 % acetic acid, and measuring their OD values at 570 nm by Multiskan Ascent. Cell invasion ability was expressed indirectly by varying OD values. HMGB1 or RAGE siRNA, HMGB1, or RAGE antibody, and sRAGE inhibited the invasion ability of HCCLM3 cells, while rhHMGB1 facilitated it (*P < 0.05, **P < 0.01). c, d Migration ability of HCCLM3 cells was detected by wound healing assay. Incubating for 0, 6, 12, and 24 h, respectively, the number of HCCLM3 cells migrating into the scraped areas was counted. (*P < 0.05, **P < 0.01)
Fig. 6
Fig. 6
Effects of HMGB1 and RAGE on NF-κB expression in HCCLM3 cells. a Expression of NF-κB p65 or p50 mRNA in HCCLM3 cells was explored by RT-PCR. b Relative expression of NF-κB p65 or p50 mRNA was normalized to β-actin. HMGB1 or RAGE siRNA, HMGB1 or RAGE antibody, and sRAGE inhibited NF-κB p65 and p50 mRNA expression in HCCLM3 cells, while rhHMGB1 increased it (* P < 0.05, ** P < 0.01). c Western blot was performed to test NF-κB p65 or p50 protein expression in HCCLM3 cells. d Quantity analysis of NF-κB p65 and p50 proteins expression levels relative to GAPDH. (* P < 0.05, ** P < 0.01)
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