Su1001 Circulating Scca-IgM Complex Is an Useful Biomarker to Monitor HCC Therapy
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Su1000 Enhanced Liver Tumorigenesis in Sulfatase 1 (SULF1) Transgenic Mice Is Associated With Activation of the Wnt-β-Catenin Signaling Pathway Chunling Hu, Shaoshan Han, Catherine D. Moser, Abdul M. Oseini, Dongye Yang, Lewis R. Roberts Background and Aims: Using in vitro cell culture and in vivo xenograft mouse models, we have previously shown that the heparan sulfate-degrading endosulfatase SULF1 functions as a tumor suppressor in established hepatocellular carcinoma cell lines by abrogating receptor tyrosine kinase signaling. In contrast, whole genome gene expression analysis of resected human HCCs showed increased expression of SULF1 in 70% of primary HCCs; and SULF1 expression in primary HCCs has been associated with activation of the oncogenic Wnt/β-catenin pathway. Here we report for the first time the results of DEN-induced hepatocarcinogenesis in a hepatocyte-specific transgenic Sulf1 mouse model in which the Sulf1 gene is driven by the transthyretin promoter. The effect of Sulf1 transgenesis on Wnt/ β-catenin activation was assessed. Methods: Liver carcinogenesis was induced in wild-type or hepatocyte-specific Sulf1 transgenic mice by intraperitoneal injection of 15 mg/kg of the liver carcinogen diethylnitrosamine (DEN) at 14 days after birth. Mice were sacrificed at 8 months of age and the harvested livers were examined for weight, tumor size and number. Cell proliferation was assessed using the BrdU proliferation index and Wnt/ β-catenin activa- tion was measured by assessment of β-catenin staining and cytoplasmic-nuclear translocation of β-catenin. Results: Consistent with the observation of increased SULF1 in primary human HCCs, Sulf1 transgenic mice showed increased liver tumorigenesis as demonstrated by tumor number, tumor volume, liver weight/body weight ratio, and BrdU proliferation index. To investigate the mechanism for this, we performed immunohistochemistry for β-catenin. Wnt-β-catenin signaling was increased in Sulf1 transgenic mice compared to wild-type mice, as assessed by both increased β-catenin immunostaining and increased cytoplasmic-nuclear translocation of β-catenin. Conclusions: In contrast to the tumor suppressor effects of SULF1 in established human cell lines, which are mediated by inhibition of the co-receptor function of heparan sulfate in receptor tyrosine kinase signaling, primary HCCs in an in vivo model of DEN-induced HCC in Sulf1 transgenic mice show increased liver tumorigenesis associated with activation of Wnt-β-catenin signaling. This suggests that the effect of Sulf1 in carcinogen- esis is context-dependent, but appears to be mediated through oncogenic Wnt- β-catenin signaling in the majority of HCCs. Su1001 Circulating Scca-IgM Complex Is an Useful Biomarker to Monitor HCC Therapy Filomena Morisco, Giovan Giuseppe Di Costanzo, Maria Guarino, Raffaella Tortora, Ilaria Loperto, Concetta Tuccillo, Carmine Ferraiuoli, Luca Beneduce, Francesco Auriemma, Nicola Caporaso Background and aims Every year hepatocellular carcinoma (HCC) develops in about 3-4% of cirrhotic patients. The serpin squamous cell carcinoma antigen (SCCA) was found elevated in liver cancer specimens and circulating SCCA-IgM complexes have been described in serum of patients with HCC. This study aimed to evaluate the ability of SCCA-IgM serum levels to monitor the efficacy of HCC therapy. Methods From April 2011 to July 2012, 106 patients (M/F 81/25, median age: 68 years, range 41-85) with a new diagnosis of HCC were enrolled in two referral centres in a prospective study. The diagnosis of HCC was made according to the AASLD 2010 guidelines. The patients were divided in two groups: Group A, including 64 patients (BCLC stage A and B) treated with loco-regional therapy (RF, LTA, PEI, TACE), and Group B including 42 patients (BCLC stage B and C) treated with Sorafenib (800mg/die). Response to therapy was evaluated with imaging techniques according to the mRECIST criteria. Serum SCCA-IgM levels were determined by the Hepa-IC kit (Xeptagen SpA, Venezia, Italy) at basal time, after 1 and 3 months from the beginning of treatment. The quantization of the complex SCCA-IgM is expressed in Arbitrary Units(AU)/ml. Results At the time of diagnosis, SCCA-IgM was reactive in 59/106 patients (56%), mean±SD: 274.4±263.2 AU/ml. In particular SCCA-IgM were positive in 34/64 (53%) patients in Group A and 25/42 (60%) patients in Group B mean±SD: 258.7±280.5 AU/ml and 295.1±242.5 AU/ml, respectively. According to mRECIST criteria, 29/34 (85%) patients in Group A and 16/25 (64%) in Group B showed a response to therapy at 1 and 3 months of follow-up, respectively. In responder patients of Group A the SCCA-IgM mean value decreases from 263,8 AU/ml at T0 to 229.7 AU/ml at T1 (p ,0,05); in responder patients of Group B it decreases from 276,7 AU/ml at T0 to 190,3 AU/ml at T3 (p ,0,001). In non-responder patients SCCA-IgM levels remain stable during the follow-up. Conclusion These results suggest that the serial determination of SCCA-IgM complex may be helpful in monitoring the outcome of therapy in patients with HCC. Su1002 Hepatocellular Carcinoma Derived Colony Stimulating Factor-1 Promotes Arginase Expression and Activity in Tumor Associated Macrophages Abdul Rehman, Priyanka Thakur, Michael Duncan Tumor-associated macrophages are an important component of the tumor microenvironment and play a crucial role in hepatocellular carcinoma (HCC) development and prognosis. Pro- inflammatory (M1-type) macrophages enhance antitumor immunity while M2-type macro- phages are immunosuppressive and promote cancer through tissue remodeling and angiogen- esis. These cells demonstrate a high degree of plasticity and can revert from one phenotype to another under appropriate stimuli. Tumor-derived factors including colony stimulating factor-1 (CSF-1) promote macrophages towards an M2 phenotype and may potentiate tumor growth and metastasis. It is well established that CSF-1 signaling is critical in the differentiation, survival and function of macrophages. The enzyme arginase is highly expressed in tumor-associated macrophages and plays a significant role in the immunosup- pressive activity of these cells by catalyzing the conversion of the amino acid L-arginine to L-ornithine and urea. We examined whether CSF-1 signaling influences arginase activity and hypothesized that CSF-1 regulates arginase expression and activity in tumor associated S-991 AASLD Abstracts macrophages. In order to test our hypothesis we developed an in vitro model of paracrine- mediated differentiation of murine bone marrow cells to macrophages. We treated murine bone marrow derived cells with condition media derived from murine hepa 1-6 HCC cell line and converted a significant fraction of these cells to f4/80 positive macrophages. These cells highly expressed M2-type markers and were largely negative for M1-type markers. Importantly, we observed a significant increase in arginase gene expression in our differen- tiated macrophage population. In order to determine the role of CSF-1 and its receptor- mediated pathways in arginase gene expression and activity, we selectively blocked CSF-1 receptor as well as the phosphatidylinositol 3-kinases (PI3K) and p38 mitogen activated kinase (p38MAPK) pathways using selective small molecule inhibitors. We observed signifi- cant decrease in arginase gene expression by quantitative real time PCR when CSF-1 receptor and the PI3K and p38MAPK pathways were inhibited. Also, an arginase enzymatic assay showed reduced activity of arginase with the inhibition of CSF-1 as well as downstream PI3K and p38MAPK signaling pathways. This data clearly demonstrates a critical role for CSF-1 signaling in arginase gene expression and enzymatic activity and provides a potential link between the CSF-1 pathway and immunosuppressive activity of tumor-associated macro- phages. Su1003 Role of Activated Hepatic Stellate Cells in Metastasis of Hepatocellular Carcinoma Nan Lin, Xu Linan Objective: The presence of liver cirrhosis is the main risk factor for the development of hepatocellular carcinoma (HCC). Activated hepatic stellate cells are recognized as a central event in the development of liver fibrosis, but which role in HCC is unclear. This study investigated angiogenetic activity of activated hepatic stellate cells in HCC and angiogenetic effect on proliferation, metastasis of HCC. Methods: In vitro, we exposured microvascular endothelial cells to conditioned media(CM) from activated hepatic stellate cells to observe the influence of the CM on microvascular endothelial cells by quantifying microvascular endothelial cell tube formation, and placed aHSC over an endothelial monolayer to assesses transendothelial migration of activated hepatic stellate cells to hepatoma cells by examining the movement of CFSE labeled activated hepatic stellate cells through the endothelial cell monolayer. For convenient viewing, microvascular endothelial cell and activated hepatic stellate cells were labeled for CFSE (one green fluorescent dye). In vivo, we established orthotopic model of HCC with nude mice receiving an intraliver injection of activated hepatic stellate cells plus hepatoma cells. After 7 weeks, tumor size, number of metastases were measured. Furthermore, tumors tissue were ashahsessed by immunostaining for expression of activated hepatic stellate cells and microvessel. Angiogenesis activities were assessed by immunostaining tumors for CD34, an endothelial cell marker and vascular endothelial growth factor (VEGF). Result: In vitro, The CM from activated hepatic stellate cells stimulated the tube formation by microvascular endothelial cell , and hepatoma cells stimulated transen- dothelial migration of activated hepatic stellate cells . In vivo, compared with mice receiving hepatoma cells alone, mice injected with hepatoma cells plus aHSC exhibited the most increased tumor size and regional and distant metastasis. CD34 and VEGF in primary tumors of mice injected with hepatoma cells plus activated hepatic stellate cells were higher expression compared to hepatoma cells alone. Activated hepatic stellate cells and microvessel were both located around nests in serial sections of HCC. The CFSE labeled activated hepatic stellate cells were found in multiple metastatic sites in each mice injected with activated hepatic stellate cells plus hepatoma cells. Conclusion: Activated hepatic stellate cells promote prolifer- ation, metastasis of HCC through angiogenesis, and spreaded to other parts of the body accompanying with HCC cells continuing with playing a role in metastatic tumor. Su1004 High Mobility Group Box-1 Can Increase miR-21 Expression in Hepatocellular Carcinoma Through STAT3 Signaling Man Chen, Ying Chang, Hai Huang, Jusheng Lin, Allan Tsung Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is associated with a very poor prognosis. The discovery of microRNA (miRNA) has significantly altered traditional concepts of gene regulation and miRNAs have emerged as novel modulators of gene expression in cancer. As an oncomir, the over-expression of miR- 21 has been reported in many cancers including HCC. However, the role and regulation of miR-21 in HCC are not elucidated. High mobility group box 1 (HMGB1) is a prototypical Damage-Associated Molecular Pattern molecule (DAMP) with multiple extracellular func- tions, including modulation of inflammation and immunity. We hypothesize that HMGB1 induces the expression of miR-21 in HCC to promote tumorigenesis. Methods: Human HCC cell lines (Huh7, HepG2, Hep3B) and normal human hepatocytes were stimulated with varying doses of recombinant HMGB1 (rHMGB1) or treated with neutralizing antibody to HMGB1. Signal transducer and activator of transcription 3 (STAT3) signaling was inhibited using the STAT3 inhibitor wp1066 to examine its role in HMGB1-mediated miR-21 induction. miR-21 expression was quantitated by Reverse transcription and TaqMan real-time polymer- ase chain reaction (Taqman qRT-PCR). Protein expressions of HMGB1, total STAT3, and STAT3 phosphorylation status at Tyr 705 (pSTAT3) were assessed by Western blot analysis. Results: miR-21, HMGB1 and pSTAT3 were all significantly up-regulated in HCC cell lines (Huh7, HepG2 and Hep3B) compared with normal hepatocytes. We also detected the significant over-expression of miR-21, HMGB1 and pSTAT3 in 20 pairs of HCC tumors compared to their matched adjacent non-tumor tissues (P .0.05). To determine if HMGB1 regulated miR-21 expression, rHMGB1 was used to stimulate the various HCC cell lines. Interestingly, miR-21 was up-regulated more than 3-fold of basal expression at 24 hours with 1ug/ml rHMGB1 stimulation. This increase of miR-21 expression was inhibited when neutralizing antibody to HMGB1 was used to treat the same cells. To determine the mecha- nism of HMGB1-mediated miR-21 regulation, we examined STAT3 signaling. Western blot analysis for pSTAT3 demonstrated that with rHMGB1 treatment, pSTAT3 expression was unregulated about 1.5-2.5 fold compared to total STAT3, while treatment with neutralizing antibody to HMGB1 inhibited the induction of pSTAT3. In addition, using the STAT3 AASLD Abstracts
Transcript of Su1001 Circulating Scca-IgM Complex Is an Useful Biomarker to Monitor HCC Therapy
Su1000
Enhanced Liver Tumorigenesis in Sulfatase 1 (SULF1) Transgenic Mice IsAssociated With Activation of the Wnt-β-Catenin Signaling PathwayChunling Hu, Shaoshan Han, Catherine D. Moser, Abdul M. Oseini, Dongye Yang, LewisR. Roberts
Background and Aims: Using in vitro cell culture and in vivo xenograft mouse models, wehave previously shown that the heparan sulfate-degrading endosulfatase SULF1 functionsas a tumor suppressor in established hepatocellular carcinoma cell lines by abrogatingreceptor tyrosine kinase signaling. In contrast, whole genome gene expression analysis ofresected human HCCs showed increased expression of SULF1 in 70% of primary HCCs;and SULF1 expression in primary HCCs has been associated with activation of the oncogenicWnt/β-catenin pathway. Here we report for the first time the results of DEN-inducedhepatocarcinogenesis in a hepatocyte-specific transgenic Sulf1 mouse model in which theSulf1 gene is driven by the transthyretin promoter. The effect of Sulf1 transgenesis on Wnt/β-catenin activation was assessed. Methods: Liver carcinogenesis was induced in wild-typeor hepatocyte-specific Sulf1 transgenic mice by intraperitoneal injection of 15 mg/kg of theliver carcinogen diethylnitrosamine (DEN) at 14 days after birth. Mice were sacrificed at 8months of age and the harvested livers were examined for weight, tumor size and number.Cell proliferation was assessed using the BrdU proliferation index and Wnt/ β-catenin activa-tion was measured by assessment of β-catenin staining and cytoplasmic-nuclear translocationof β-catenin. Results: Consistent with the observation of increased SULF1 in primary humanHCCs, Sulf1 transgenic mice showed increased liver tumorigenesis as demonstrated by tumornumber, tumor volume, liver weight/body weight ratio, and BrdU proliferation index. Toinvestigate the mechanism for this, we performed immunohistochemistry for β-catenin.Wnt-β-catenin signaling was increased in Sulf1 transgenic mice compared to wild-type mice,as assessed by both increased β-catenin immunostaining and increased cytoplasmic-nucleartranslocation of β-catenin. Conclusions: In contrast to the tumor suppressor effects of SULF1in established human cell lines, which are mediated by inhibition of the co-receptor functionof heparan sulfate in receptor tyrosine kinase signaling, primary HCCs in an in vivo modelof DEN-induced HCC in Sulf1 transgenic mice show increased liver tumorigenesis associatedwith activation ofWnt-β-catenin signaling. This suggests that the effect of Sulf1 in carcinogen-esis is context-dependent, but appears to be mediated through oncogenic Wnt- β-cateninsignaling in the majority of HCCs.
Su1001
Circulating Scca-IgM Complex Is an Useful Biomarker to Monitor HCCTherapyFilomena Morisco, Giovan Giuseppe Di Costanzo, Maria Guarino, Raffaella Tortora, IlariaLoperto, Concetta Tuccillo, Carmine Ferraiuoli, Luca Beneduce, Francesco Auriemma,Nicola Caporaso
Background and aims Every year hepatocellular carcinoma (HCC) develops in about 3-4%of cirrhotic patients. The serpin squamous cell carcinoma antigen (SCCA) was found elevatedin liver cancer specimens and circulating SCCA-IgM complexes have been described inserum of patients with HCC. This study aimed to evaluate the ability of SCCA-IgM serumlevels to monitor the efficacy of HCC therapy. Methods From April 2011 to July 2012, 106patients (M/F 81/25, median age: 68 years, range 41-85) with a new diagnosis of HCC wereenrolled in two referral centres in a prospective study. The diagnosis of HCC was madeaccording to the AASLD 2010 guidelines. The patients were divided in two groups: GroupA, including 64 patients (BCLC stage A and B) treated with loco-regional therapy (RF, LTA,PEI, TACE), and Group B including 42 patients (BCLC stage B and C) treated with Sorafenib(800mg/die). Response to therapy was evaluated with imaging techniques according to themRECIST criteria. Serum SCCA-IgM levels were determined by the Hepa-IC kit (XeptagenSpA, Venezia, Italy) at basal time, after 1 and 3 months from the beginning of treatment.The quantization of the complex SCCA-IgM is expressed in Arbitrary Units(AU)/ml. ResultsAt the time of diagnosis, SCCA-IgM was reactive in 59/106 patients (56%), mean±SD:274.4±263.2 AU/ml. In particular SCCA-IgM were positive in 34/64 (53%) patients in GroupA and 25/42 (60%) patients in Group B mean±SD: 258.7±280.5 AU/ml and 295.1±242.5AU/ml, respectively. According to mRECIST criteria, 29/34 (85%) patients in Group A and16/25 (64%) in Group B showed a response to therapy at 1 and 3 months of follow-up,respectively. In responder patients of Group A the SCCA-IgM mean value decreases from263,8 AU/ml at T0 to 229.7 AU/ml at T1 (p ,0,05); in responder patients of Group B itdecreases from 276,7 AU/ml at T0 to 190,3 AU/ml at T3 (p ,0,001). In non-responderpatients SCCA-IgM levels remain stable during the follow-up. Conclusion These resultssuggest that the serial determination of SCCA-IgM complex may be helpful in monitoringthe outcome of therapy in patients with HCC.
Su1002
Hepatocellular Carcinoma Derived Colony Stimulating Factor-1 PromotesArginase Expression and Activity in Tumor Associated MacrophagesAbdul Rehman, Priyanka Thakur, Michael Duncan
Tumor-associated macrophages are an important component of the tumor microenvironmentand play a crucial role in hepatocellular carcinoma (HCC) development and prognosis. Pro-inflammatory (M1-type) macrophages enhance antitumor immunity while M2-type macro-phages are immunosuppressive and promote cancer through tissue remodeling and angiogen-esis. These cells demonstrate a high degree of plasticity and can revert from one phenotypeto another under appropriate stimuli. Tumor-derived factors including colony stimulatingfactor-1 (CSF-1) promote macrophages towards an M2 phenotype and may potentiatetumor growth and metastasis. It is well established that CSF-1 signaling is critical in thedifferentiation, survival and function of macrophages. The enzyme arginase is highlyexpressed in tumor-associated macrophages and plays a significant role in the immunosup-pressive activity of these cells by catalyzing the conversion of the amino acid L-arginine toL-ornithine and urea. We examined whether CSF-1 signaling influences arginase activityand hypothesized that CSF-1 regulates arginase expression and activity in tumor associated
S-991 AASLD Abstracts
macrophages. In order to test our hypothesis we developed an in vitro model of paracrine-mediated differentiation of murine bone marrow cells to macrophages. We treated murinebone marrow derived cells with condition media derived from murine hepa 1-6 HCC cellline and converted a significant fraction of these cells to f4/80 positive macrophages. Thesecells highly expressed M2-type markers and were largely negative for M1-type markers.Importantly, we observed a significant increase in arginase gene expression in our differen-tiated macrophage population. In order to determine the role of CSF-1 and its receptor-mediated pathways in arginase gene expression and activity, we selectively blocked CSF-1receptor as well as the phosphatidylinositol 3-kinases (PI3K) and p38 mitogen activatedkinase (p38MAPK) pathways using selective small molecule inhibitors. We observed signifi-cant decrease in arginase gene expression by quantitative real time PCR when CSF-1 receptorand the PI3K and p38MAPK pathways were inhibited. Also, an arginase enzymatic assayshowed reduced activity of arginase with the inhibition of CSF-1 as well as downstreamPI3K and p38MAPK signaling pathways. This data clearly demonstrates a critical role forCSF-1 signaling in arginase gene expression and enzymatic activity and provides a potentiallink between the CSF-1 pathway and immunosuppressive activity of tumor-associated macro-phages.
Su1003
Role of Activated Hepatic Stellate Cells in Metastasis of HepatocellularCarcinomaNan Lin, Xu Linan
Objective: The presence of liver cirrhosis is the main risk factor for the development ofhepatocellular carcinoma (HCC). Activated hepatic stellate cells are recognized as a centralevent in the development of liver fibrosis, but which role in HCC is unclear. This studyinvestigated angiogenetic activity of activated hepatic stellate cells in HCC and angiogeneticeffect on proliferation, metastasis of HCC. Methods: In vitro, we exposured microvascularendothelial cells to conditioned media(CM) from activated hepatic stellate cells to observethe influence of the CM on microvascular endothelial cells by quantifying microvascularendothelial cell tube formation, and placed aHSC over an endothelial monolayer to assessestransendothelial migration of activated hepatic stellate cells to hepatoma cells by examiningthe movement of CFSE labeled activated hepatic stellate cells through the endothelial cellmonolayer. For convenient viewing, microvascular endothelial cell and activated hepaticstellate cells were labeled for CFSE (one green fluorescent dye). In vivo, we establishedorthotopic model of HCC with nude mice receiving an intraliver injection of activated hepaticstellate cells plus hepatoma cells. After 7 weeks, tumor size, number of metastases weremeasured. Furthermore, tumors tissue were ashahsessed by immunostaining for expressionof activated hepatic stellate cells and microvessel. Angiogenesis activities were assessed byimmunostaining tumors for CD34, an endothelial cell marker and vascular endothelialgrowth factor (VEGF). Result: In vitro, The CM from activated hepatic stellate cells stimulatedthe tube formation by microvascular endothelial cell , and hepatoma cells stimulated transen-dothelial migration of activated hepatic stellate cells . In vivo, compared with mice receivinghepatoma cells alone, mice injected with hepatoma cells plus aHSC exhibited the mostincreased tumor size and regional and distant metastasis. CD34 and VEGF in primary tumorsofmice injectedwith hepatoma cells plus activated hepatic stellate cells were higher expressioncompared to hepatoma cells alone. Activated hepatic stellate cells and microvessel were bothlocated around nests in serial sections of HCC. The CFSE labeled activated hepatic stellatecells were found in multiple metastatic sites in each mice injected with activated hepaticstellate cells plus hepatoma cells. Conclusion: Activated hepatic stellate cells promote prolifer-ation, metastasis of HCC through angiogenesis, and spreaded to other parts of the bodyaccompanying with HCC cells continuing with playing a role in metastatic tumor.
Su1004
High Mobility Group Box-1 Can Increase miR-21 Expression in HepatocellularCarcinoma Through STAT3 SignalingMan Chen, Ying Chang, Hai Huang, Jusheng Lin, Allan Tsung
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwideand is associated with a very poor prognosis. The discovery of microRNA (miRNA) hassignificantly altered traditional concepts of gene regulation and miRNAs have emerged asnovel modulators of gene expression in cancer. As an oncomir, the over-expression of miR-21 has been reported in many cancers including HCC. However, the role and regulationof miR-21 in HCC are not elucidated. High mobility group box 1 (HMGB1) is a prototypicalDamage-Associated Molecular Pattern molecule (DAMP) with multiple extracellular func-tions, including modulation of inflammation and immunity. We hypothesize that HMGB1induces the expression of miR-21 in HCC to promote tumorigenesis. Methods: Human HCCcell lines (Huh7, HepG2, Hep3B) and normal human hepatocytes were stimulated withvarying doses of recombinant HMGB1 (rHMGB1) or treated with neutralizing antibody toHMGB1. Signal transducer and activator of transcription 3 (STAT3) signaling was inhibitedusing the STAT3 inhibitor wp1066 to examine its role inHMGB1-mediatedmiR-21 induction.miR-21 expression was quantitated by Reverse transcription and TaqMan real-time polymer-ase chain reaction (Taqman qRT-PCR). Protein expressions of HMGB1, total STAT3, andSTAT3 phosphorylation status at Tyr 705 (pSTAT3) were assessed by Western blot analysis.Results: miR-21, HMGB1 and pSTAT3 were all significantly up-regulated in HCC cell lines(Huh7, HepG2 and Hep3B) compared with normal hepatocytes. We also detected thesignificant over-expression of miR-21, HMGB1 and pSTAT3 in 20 pairs of HCC tumorscompared to their matched adjacent non-tumor tissues (P.0.05). To determine if HMGB1regulated miR-21 expression, rHMGB1 was used to stimulate the various HCC cell lines.Interestingly, miR-21 was up-regulated more than 3-fold of basal expression at 24 hourswith 1ug/ml rHMGB1 stimulation. This increase of miR-21 expression was inhibited whenneutralizing antibody to HMGB1 was used to treat the same cells. To determine the mecha-nism of HMGB1-mediated miR-21 regulation, we examined STAT3 signaling. Western blotanalysis for pSTAT3 demonstrated that with rHMGB1 treatment, pSTAT3 expression wasunregulated about 1.5-2.5 fold compared to total STAT3, while treatment with neutralizingantibody to HMGB1 inhibited the induction of pSTAT3. In addition, using the STAT3
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