- Email: [email protected]
FOXO3a PATHWAY
POSTERS expression of microRNA let-7a was found to deregulate ~200 genes involved in cell proliferation, differentiation and adhesion. Among them is the Insulin-like Growth Factor-II (IGF-II) which is involved in the pathogenesis of Hepatocellular carcinoma (HCC). The embedment of the let-7a-3 gene in a well defined CpG island on chromosome 22q12.31 suggests that its expression might be epigenetically controlled via DNA methylation. The impact of DNA methylation on the expression of the primary and mature let-7a-3 transcripts has never been investigated in HCC. Thus, we aimed at investigating whether DNA methylation is responsible for let-7a downregulation and consequently on IGF-II expression in HCC. Methods: Total RNAs were isolated from 16 HCV-induced HCC tissues and 9 healthy liver tissues. Expression of both Primary and mature microRNA let-7a as well as the IGF-II mRNA was analyzed using RT-qPCR. In addition, DNA extracted from HCC tissues was bisulfite converted and its methylation status was assessed by PCR using two sets of nested primers to amplify the whole CpG Island followed by quantification by SYBR-Green qPCR using methylation and non-methylation specific sets of primers. Results: Bioinformatics analysis revealed three possible hits where let-7a may target the IGF-II 3 UTR with promising scores. Also, the expression of primary and mature microRNA let-7a showed significant downregulation in HCC tissues compared to healthy tissues (p = 0.0203 and p = 0.0208, respectively). Furthermore, we found a significant inverse correlation between mature let-7a and IGF-II mRNA expression (Pearson R = −0.21). Interestingly, the methylation analysis showed hypermethylation (88%) of the investigated CpG island in the let-7a-3 gene. Conclusion: For the first time, we showed that hypermethylation of micro-RNA let-7a-3 gene is responsible for the downregulation of both the primary and mature transcripts which lead to an exaggerated IGF-II mitogenic signal contributing to malignant transformation. 1098 TLR4 DEFICIENCY PROTECTS AGAINST HCC INITIATION IN A FIBROTIC LIVER S.N. Weber, A. Bohner, F. Lammert. Department of Medicine II, Saarland University Medical Center, Homburg, Germany E-mail: [email protected] Background: The development of hepatocellular carcinoma (HCC) is usually the consequence of advanced liver fibrosis but the mechanisms are still poorly understood. Recently it has been shown that HCC promotion depends on Toll-like receptor (Tlr) 4 and intestinal microbiotia (Dapito et al. Cell 2012). HCC initiation can be modelled in mice by the administration of diethylnitrosamine (DEN), with HCC formation depending on interleukin (IL) 6 production (Naugler et al. Science 2007). Mice that lack the hepatocanalicular phosphatidylcholine transporter Abcb4 develop biliary fibrosis and can be used as a model to study tumor formation in injured liver. The aim of our study was to investigate HCC initiation in Abcb4-deficient (Abcb4−/− Tlr4+/+ ) and Abcb4/Tlr4double-deficient (Abcb4−/− Tlr4−/−) mice. Methods: Abcb4-deficient mice on the FVB/NJ genetic background were crossed to two distinct genetic backgrounds (Tlr4-sufficient C3H/HeN and Tlr4-deficient C3H/HeJ) for at least 10 generations. Hepatic collagen contents were evaluated by hydroxyproline (Hyp) assay at the age of 16 weeks (when biliary fibrosis is established). Congenic knockout and wild-type (wt) mice were treated with a single dose of DEN. Phenotypic differences after 48 hours were determined by analyzing hepatic apoptosis (TUNEL) and proliferation (Ki67) rates as well as inflammatory markers including IL6 expression. Results: Hepatic collagen contents were significantly reduced in Abcb4−/− Tlr4−/− as compared to Abcb4−/− Tlr4+/+ mice (282.84±18.21 vs. 327.90±13.61 mg Hyp/g liver). After DEN challenge, apoptosis, proliferation and inflammation (IL6 expression) were significantly
(p < 0.05) increased in Abcb4-deficient mice as compared to controls. Of note, proliferation rates were significantly (p < 0.05) lower in Abcb4−/− Tlr4−/− mice (6.43±0.61 vs. 10.96±1.05% proliferative cells), IL6 expression was markedly reduced (44.42±9.19 vs. 63.29±7.32-fold expression, untreated C3H/HeN wt was set to 1.00) and apoptosis was slightly elevated (7.07±0.91 vs. 6.00±0.82% apoptotic cells). Discussion: This study demonstrates that HCC initiation upon DEN challenge depends on pre-existing fibrosis and genetic background. Our findings indicate that Tlr4 deficiency protects from HCC initiation in Abcb4/Tlr4-double-deficient mice. 1099 MESENCHYMAL STEM CELLS MODIFIED TO EXPRESS INTERFERON-b INHIBIT THE GROWTH OF HEPATOCELLULAR CANCER THROUGH INHIBITING AKT/FOXO3a PATHWAY C. Xie, J.-Q. Xie, Q.-S. Zhang, L. Peng, G.-L. Zhang, D.-Y. Xie, Z.-L. Gao. Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China E-mail: [email protected] Objective: This study aim to investigate using of bone marrow mesenchymal stem cells (BMSC) genetically engineered to produce interferon-b (IFN-b) as a gene delivery system to treat liver cancer in vitro and in vivo. Methods: To measure the effects on tumor cell growth, IFN-bproducing BMSCs (BMSCs/IFN-b) were established. We used ELISA to detect the IFN-b secretion in the BMSC culture condition medium (CM) and measured the effect of BMSCs/IFN-b on hepatoma cells proliferation by MTT and colony formation assay. We used Brdu staining and cell cycle analysis to investigate the effect of BMSC on hepatoma cell cycle. RT-PCR and Western blotting was used to detect cell cycle related proteins and AKT signaling pathway. Results: BMSCs/IFN-b cells can stably secrete high levels of IFN-b. MTT showed hepatoma cell had a lower growth rate from the first three days when cultured in BMSC/IFN-b-CM compared to in BMSC/vector-CM or DMEM culture group. The number of colony forming and clone size detected in BMSC/IFN-b-CM cultured cells was less than the control group. Co-cultured with BMSC/IFN-b-CM dramatically decreased the percentages of cells with incorporated BrdUrd. In BMSC/IFN-b-CM treated liver cancer cells, the proportion of G1 phase cells increased but decreased in S phase of the cell (the ratio for BMSC/vector: BMSC/IFN-b in HepG2: 20.5±1.8%: 11.2±1.2%, t = 7.53, P < 0.01; Huh7: 24.7±1.8%: 12.7±2.0%, t = 7.87, P < 0.01). BMSC/IFN-b inhibited HCC growth in NID/SCID mouse. Compared with the control group, P21 and P27 expression of hepatoma cells increased, while CyclinD1 and phosphorylation of Rb expression decreased when co-cultured with BMSC/IFN-b-CM. It was mechanistically associated with suppression of Akt activity and enhanced transcriptional activity of FOXO3a. Conclusion: IFN-b gene modified BMSC can effectively inhibit the proliferation of hepatoma cells in vitro and in vivo through inhibit AKT/FOXO3a pathway. These results indicate that IFN-b/BMSCs are a powerful anti-cancer cytotherapeutic tool for HCC. 1100 REGULATION OF RADIATION INDUCED LIVER CANCER STEM CELL MIGRATION AND METASTASIS BY ADAM17 S.K. Yoon, S.W. Hong, W. Hur, Y.K. Lee, S.W. Choi, S.H. Cho, J.M. Yang, Y.S. Lee, C.D. Lee. WHO Collaborating Center of Viral Hepatitis and Department of Internal Medicine College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea E-mail: [email protected] Background and Aims: Recent studies have described that cancer stem cell plays a key role radioresistance. The cell surface marker CD133 has been known as a cancer stem cell marker expressed in hepatocellular carcinoma (HCC). ADAM17 gene was reported
Journal of Hepatology 2013 vol. 58 | S409–S566
S449
(p < 0.05) increased in Abcb4-deficient mice as compared to controls. Of note, proliferation rates were significantly (p < 0.05) lower in Abcb4−/− Tlr4−/− mice (6.43±0.61 vs. 10.96±1.05% proliferative cells), IL6 expression was markedly reduced (44.42±9.19 vs. 63.29±7.32-fold expression, untreated C3H/HeN wt was set to 1.00) and apoptosis was slightly elevated (7.07±0.91 vs. 6.00±0.82% apoptotic cells). Discussion: This study demonstrates that HCC initiation upon DEN challenge depends on pre-existing fibrosis and genetic background. Our findings indicate that Tlr4 deficiency protects from HCC initiation in Abcb4/Tlr4-double-deficient mice. 1099 MESENCHYMAL STEM CELLS MODIFIED TO EXPRESS INTERFERON-b INHIBIT THE GROWTH OF HEPATOCELLULAR CANCER THROUGH INHIBITING AKT/FOXO3a PATHWAY C. Xie, J.-Q. Xie, Q.-S. Zhang, L. Peng, G.-L. Zhang, D.-Y. Xie, Z.-L. Gao. Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China E-mail: [email protected] Objective: This study aim to investigate using of bone marrow mesenchymal stem cells (BMSC) genetically engineered to produce interferon-b (IFN-b) as a gene delivery system to treat liver cancer in vitro and in vivo. Methods: To measure the effects on tumor cell growth, IFN-bproducing BMSCs (BMSCs/IFN-b) were established. We used ELISA to detect the IFN-b secretion in the BMSC culture condition medium (CM) and measured the effect of BMSCs/IFN-b on hepatoma cells proliferation by MTT and colony formation assay. We used Brdu staining and cell cycle analysis to investigate the effect of BMSC on hepatoma cell cycle. RT-PCR and Western blotting was used to detect cell cycle related proteins and AKT signaling pathway. Results: BMSCs/IFN-b cells can stably secrete high levels of IFN-b. MTT showed hepatoma cell had a lower growth rate from the first three days when cultured in BMSC/IFN-b-CM compared to in BMSC/vector-CM or DMEM culture group. The number of colony forming and clone size detected in BMSC/IFN-b-CM cultured cells was less than the control group. Co-cultured with BMSC/IFN-b-CM dramatically decreased the percentages of cells with incorporated BrdUrd. In BMSC/IFN-b-CM treated liver cancer cells, the proportion of G1 phase cells increased but decreased in S phase of the cell (the ratio for BMSC/vector: BMSC/IFN-b in HepG2: 20.5±1.8%: 11.2±1.2%, t = 7.53, P < 0.01; Huh7: 24.7±1.8%: 12.7±2.0%, t = 7.87, P < 0.01). BMSC/IFN-b inhibited HCC growth in NID/SCID mouse. Compared with the control group, P21 and P27 expression of hepatoma cells increased, while CyclinD1 and phosphorylation of Rb expression decreased when co-cultured with BMSC/IFN-b-CM. It was mechanistically associated with suppression of Akt activity and enhanced transcriptional activity of FOXO3a. Conclusion: IFN-b gene modified BMSC can effectively inhibit the proliferation of hepatoma cells in vitro and in vivo through inhibit AKT/FOXO3a pathway. These results indicate that IFN-b/BMSCs are a powerful anti-cancer cytotherapeutic tool for HCC. 1100 REGULATION OF RADIATION INDUCED LIVER CANCER STEM CELL MIGRATION AND METASTASIS BY ADAM17 S.K. Yoon, S.W. Hong, W. Hur, Y.K. Lee, S.W. Choi, S.H. Cho, J.M. Yang, Y.S. Lee, C.D. Lee. WHO Collaborating Center of Viral Hepatitis and Department of Internal Medicine College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea E-mail: [email protected] Background and Aims: Recent studies have described that cancer stem cell plays a key role radioresistance. The cell surface marker CD133 has been known as a cancer stem cell marker expressed in hepatocellular carcinoma (HCC). ADAM17 gene was reported
Journal of Hepatology 2013 vol. 58 | S409–S566
S449