Liver Cancer Development Process In The Transcriptional Regulation Of Angiogenic Factors Nogob Mechanism

Hepatocarcinoma (HCC) is one of the common malignant tumors in China.110 thousands of people died of HCC every year. NogoB, also known as RTN4B, was cloned by our lab in 1998. It is a member of reticulon (RTN) protein family which is localized in the endoplasmic reticulum. It is found that NogoB plays dual roles in both nerve and muscle system. It is also reported that NogoB takes part in vascular remodeling by promoting migration and adhesion of vascular endothelial cells. However, our group found that NogoB is expressed highly and seretedly in liver cancer compared to its expression in adjacent tumor tissue. So we presumed and confirmed in the former studies that highly expressed NogoB plays positive roles in tumor angiogenesis, and then promotes tumorigenesis in HCC. To explore the reason of the specific characterization of NogoB in HCC, we worked on its mechanism of transcriptional regulation.Along with the uncontrolled proliferation, tumor cells that were located more than 180μm away from blood vessels were observed to necrose. This is similar to the distance that oxygen diffuses as it passes from the capillary to cells. In response to the hypoxia condition, cancer cells undergo genetic changes to induce new blood vessel to provide rapidly proliferating tumor cells with an adequate supply of oxygen and metabolites. Hypoxia inducible factor (HIF)-la is one of the key regulator in hypoxia induced angiogenesis. HIF-1a is a transcription factor widely expressed in human and mammalian. Its multiple target genes have been proved to modulate angiogenesis by promoting proliferation and migration in endothelial cells. Based on the evidence, we speculated that the overexpression of novel pro-angiogenesis factor NogoB is also regulated by HIF-la in HCC.By comparing the expression patterns of NogoB in HCC and its normal counterpart by Western blot analyses, we found that both NogoB and HIF-la was significantly up-regulated in HCC samples in comparison to the adjacent non-tumorous liver tissues. Then we use rat hepatic artery ligation (HAL) model to make the ligated liver physiological hypoxia, and this induced the expression of NogoB. Deferoxamine (DFO) and CoCl2 could mimic hypoxia conditions by means of stabilizing HIF-1α. In CoCl2 treated rat primary liver cells, increased expression of NogoB was also observed. By using hepatocarcinoma cell lines as a model, we found that in response to DFO or hypoxic condition, the expression of NogoB increased in a time-dependent manner. To determine whether elevated expression of NogoB was mediated by HIF-la, we utilized luciferase assay. The result showed that transfected HIF-la . increased the NogoB promoter activity in a dose-dependent manner and lentivirus delivery of HIF-1a siRNA reduced the NogoB promoter activity in hypoxia condition. Analysis of the NogoB promoter showed two predicted hypoxia response elements (HRE). To identify the HIF-la binding site in NogoB promoter, a series of NogoB promoter deletion constructs was generated. The promoter region responsible to hypoxia span from-4041 to-991 relative to transcription start site. Then a site-directed mutant was constructed at putative HRE located-3829 to-3822 upstream of transcription start site. No significant increase of NogoB promoter activity was detected either in hypoxia or in DFO treated culture. Chromatin immunoprecipitation assay (ChIP) proved that HIF-la protein is bound to the distal HRE. A gel mobility shift assay confirmed that the interaction between HIF-1 and the HRE in the NogoB promoter is direct. Our observation suggeted that hypoxia may play a central role in NogoB upregulation in HCC and further elucidate the molecular mechanisms responsible for hypoxia mediated induction of NogoB expression in HCC cells.That hypoxia strongly induced NogoB is an important mechanism of induction in tumor. However, many tumor-associatied angiogenetic factors could also be elevated by other transcriptional factors in response to diverse simuli. The AP-1 (activator protein 1) transcription factor, known as basic leucine-zipper (bZIP) protein, is a dimeric complex which is composed of Jun, Fos, ATF and Maf protein families. The Jun proteins could form homodimers or heterodimers with Fos proteins which dimerize with Jun proteins to enhance their DNA-binding activity. By binding to the TPA response element (TRE) in the promoter, AP-1 complex transcriptional regulates the expression of target genes and thereby plays a role in the proliferation, invasion, metastasis, transformation and angiogenesis of tumors. Our data indicated both NogoB and AP-1 was significantly up-regulated in HCC samples in comparison to the adjacent non-tumorous liver tissues. Transient expression of AP-1 and activation of endogenous AP-1 by treatment with TPA increased the NogoB expression and its promoter activity. And the induction of NogoB promoter activity was mediated by c-Fos but not c-Jun. A-Fos, a dominant negative construct of AP-1, showed to inhibit AP-1-dependent and TPA induced NogoB promoter activity. The A-Fos was able to prevent AP-1 induction of the promoter activity, but only slightly decreased the basal activity of the promoter. This indicated that constitutive NogoB expression is only slightly due to the effect of AP-1. Bioinformatic analysis revealed three putative AP-1 response elements. Sequential deletion of the promoter revealed that the region located upstream of transcription initiation site (-991/-770) was essential for AP-1 induction of NogoB transcription. Site-directed mutation analysis of three potential AP-1 binding sites suggested that the distal element at-782bp to-776bp was responsible for the induction, which was confirmed by chromatin immunoprecipitation.In conclusion, our results provided the molecular basis that the overexpression of NogoB was mainly induced by HIF-la in hypoxic condition. Meanwhile AP-1 also played an important role in regulation of NogoB in response to diverse simuli.