| Hepatocellular carcinoma (HCC) is one of the most prevalent cancers with high fatality, which is the third highest cancer killer in the world, and is ranked second in China. The overall survival of patients with HCC is still unsatisfactory. Surgery remains the best treatment for a curative outcome of HCC, however, metastasis and recurrence is the major obstacle for further improve survival after surgery. Metastasis is a multigene-involved, multistep, and changing process, and regulated by various signaling pathways and transcription factors. Recently, overactivity and high expression of some transcription factors were found in nuclei of some malignant tumor cells, and act as modulators of cell proliferation, invasion, neo-angiogenesis, and metastasis. There are growing evidences show that transcription factors play a critical role in the invasion and metastasis of tumors by regulating transcription and expression of many metastasis associated genes. However, most of previous studies on transcription factor were focused primarily on protein and mRNA expression, and high-throughput functional analysis of multiple transcription factors was rarely reported. A novel technology - Protein/DNA array allows to profile the DNA binding activity of multiple transcription factors in a single array experiment, which make high-throughput functional analysis of multiple transcription factors possible. The aim of this study was to examine the activities of transcription factors in human HCC cell lines with different metastatic potentials, so as to identify transcription factors associated with HCC metastasis, and explore new predictive biomarkers and therapeutic targets.Interferon alpha (IFN-α) is one of few effective agents clinically used to prevent tumor metastasis. Previous studies from our institution found that IFN-α treatment dose dependently inhibits tumor growth and metastasis in metastatic nude mice model mediated by inhibition of VEGF gene transcription and expression, and resulted in inhibition of angiogenesis. However, the underlying mechanism remains unclear. Theaim of this study was to analyze the transcriptional mechanism of IFN-α on VEGF expression, angiogenesis and metastasis. So Sp1 plays a crucial role in the antiangiogenesis effect of IFN α, and may be a predictive biobarker of response of HCC patients to IFNα.Part oneTranscription factor activity profile of human hepatocellular carcinoma cell lines with different metastatic potentials and functional analysis of differential transcription factorsIn our institution, the establishment of a human HCC metastasis model system with similar genetic background and different metastatic potentials has provided a good platform for this study. This part is aimed to examine the activities of transcription factors in human HCC cell lines with different metastatic potentials, so as to identify transcription factors associated with HCC metastasis and analyze the biological activity of different transcription factors. Using Protein/DNA array, transcription factor activity profile of Hep3B, MHCC97L and MHCC97H, three HCC cell lines with different metastatic potentials, were examined. Seven activity differential transcription factors were found, 5 showed increased activity including p53, hypoxia inducible factor-1 alpha (HIF-1α), signal transducer and activator of transcription 3 (Stat3) and Sp1, and 2 showed decreased activity including Rb and Smad3. Transcription factor Sp1 is associated with HCC metastasis and being confirmed by electrophoretic mobility shift assays (EMSA). Using western blot and real-time PCR, We detected Sp1 expression in transcription, translation and post-translation modification (phosphorylation, et al) level, and found that all of mRNA, protein expression, and phosphorylation status of Sp1 contributed to overactivity of Sp1 and also positively correlated to metastatic potential of HCC cells. Using RNA interference technique, we could specifically reduce expression level and DNA binding activity of Sp1 in MHCC97H cells (with high metastatic potential), and alteration of biological behavior of MHCC97H was found. MTT test showed that after transfecting of Sp1 siRNA, significant change of viability of MHCC97H cells was not observed. However, cell invasion assay in vitro showed that the decreased expression andactivity of Sp1 can significant decrease the invasiveness of MHCC97H cells.Part twoThe mechanism of the effect of interferon alpha on VEGF transcription and angiogenesisAs had mentioned, interferon alpha (IFN-α) can decrease vescular endothelial growth factor(VEGF) gene transcription and expression, and inhibit angiogenesis and metastasis. To elucidate the underlying transcriptional mechanism, this part will firstly determine the critical regions on VEGF responsible for the transcriptional inhibitory by IFN-α. A series of 5' deletion mutants of VEGF promoter reporter gene based on the 2274-bp VEGF promoter were transfected into MHCC97H cells, and the effect of IFN-α on the VEGF promoter activity was examined. The result showed that the -109/-61 region contains essential regulatory elements. Secondly, to determine which transcription factor binding sites play the crucial role in regulation of VEGF promoter activity. The results showed that mutation in four Sp1 sites eliminated the inhibitory effect of IFN-α on VEGF promoter activity, whereas such effect was not found in mutation of either AP-2 or Egr-1 sites. It is suggested that Sp1 binding sites in this region are responsible for IFN-α-mediated inhibition of VEGF promoter activity. Thirdly, to determine whether inhibition of VEGF promoter activity by IFN-α is caused by changes in the interaction between nuclear protein and the promoter, protein/DNA array was performed using oligonucleotides corresponding to VEGF promoter as probe. It is demonstrated that IFN-α reduced the binding activity of 9 transcription factors, but only Sp1 has binding site in the -109/-61 region. EMS was performed using Sp1 consensus sequence and oligonucleotides corresponding to VEGF promoter region of nt -109 to-61, and found that Sp1 can bind to the region, and IFN-α treatment significantly reduced the binding activity of Sp1. Fourthly, to determine the role of Sp1 in inhibition of VEGF promoter activity by IFN-α, using RNA interference technique to reduce expression level and DNA binding activity of Sp1 in MHCC97H, and found that the inhibitory effect of IFN-α on VEGF promoter activity was almost eliminated. We then investigated whether the attenuation of Sp1 binding caused by IFN-α was due to decreased Sp1 protein expression and/orposttranslation modification. Western blot was performed, and found that IFN-α reduced both Sp1 protein level and Sp1 phosphorylation and decreased its DNA binding and transactivation activity.Conclusions1. There are some activity differential transcription factors in hepatocellular carcinoma cells with different metastatic potential.2. The activity and protein expression of transcription factor Sp1 is associated with metastatic potential of hepatocellular carcinoma.3. The inhibition effect of IFNα on VEGF-mediated tumor angiogenesis and metastasis is associated with the alteration of activity and protein expression of Spl.The potential application of this work1. The transcription factors, identified to associate with HCC metastasis, could be helpful to expand our understanding on mechanism of HCC metastasis and identify new predictive biomarkers and therapeutic targets.2. The association of transcription factor Sp1 with metastatic potential of hepatocellular carcinoma indicates that Sp1 might be a potential predictive biomarkers and therapeutic targets of HCC metastasis.3. The demonstration that IFN-α inhibits VEGF transcription mediated by downregulation of Sp1 could be of impact in patient selection, elucidation of drug resistance and designing combination treatment for using IFN-α to prevent HCC metastasis.The novelty of this work1. Demonstrate the transcription factor activity profile of human hepatocellular carcinoma cell lines with different metastatic potentials, and the association of Sp1 with HCC metastasis.2. Explore the mechanism of inhibitory effect of interferon alpha on VEGF transcription in HCC cell line.
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