The Expression, Functions And Mechanisms Of Neuroglobin In Hepatocellular Carcinoma

Liver cancer is the sixth most common malignancy and the third leading cause of cancer-related mortality worldwide. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults. In most cases, hepatocellular carcinoma develops within an existing background of chronic liver diseases, particularly liver cirrhosis. The risk factors of HCC include chronic hepatitis virus infection, aflatoxin B1 intake, alcohol abuse and non-alcoholic fatty liver disease, etc. However, the mechanisms of HCC are unclear. The potential molecular mechanisms include proto-oncogenes activation, tumor-suppressor genes inactivation, DNA abnormality and chromosomal instability. In addition, Oxidative stress as an independent etiological factor in the process of HCC development had been well documented.Neuroglobin (Ngb) is a novel discovered intracellular respiratory protein of haemoglobin family. It may serves as an endogenous ROS scavenger in protecting nerve cells from hypoxia and ischema injury. However, some studies indicated that Ngb is associated with tumor development, the exact function and mechanism of Ngb in tumors remains unclear.To investigate the roles and the potential mechanisms of neuroglobin on HCC development, surgically resected human tumor tissues, HCC cell line and nude mouse were applied. Respectively, cell culture, molecular biology technology, flow cytometry analysis and histomorphological methods were performed for this study. Our major findings are1) Ngb is specifically deregulated in HCC tissues.Immunohistochemistry (IHC) evaluated Ngb expression in 6 human common tumor tissues, the result showed that Ngb is specifically deregulated in HCC tissues, as compared with adjacent non-tumor tissues. Real time RT-PCR revealed that Ngb mRNA expression level is also deregulated in HCC tissues compared to the level in adjacent non-tumor tissues.2) The ectopic expression of Ngb attenuates the viability, colony formation in soft agar of HCC cells.3) Ngb knockdown promotes the viability, colony formation in soft agar of HCC cells. MTT assay and colony formation in soft agar revealed that Ngb expression/knockdown suppresses/promotes the viability and colony formation of HCC cells, as compared to the control of empty vector.4)The ectopic Ngb expression suppresses the tumorigenicity of HCC cells, whereas absence of Ngb promotes the tumorigenicity of HCC cells in vivo.5) Ngb suppresses the proliferation and the G0/G1-S transition of HCC cells via deregulating Raf-1/MAPK signal pathway.Firstly, EdU incorporation assay and flow cytometry analysis revealed that the proliferation and G0/G1-S transition of HCC cells are down-/up-regulated in presence/absence Ngb expression of HCC cells, meanwhile, immunobloting evaluated the activity of Raf-1/MAPK cascades, the result showed that Ngb deregulates the activity of this cascades, whereas absence of Ngb expression enhances its'activity. The further study indicated that the blockade of Raf-1/MAPK cascade activity removes the inhibition of Ngb on the proliferation of HCC.6) Ngb regulates the proliferation of HCC via coupling oxidative stress signalReduced GSH is one of the most important intracellular anti-oxidant agent, it may indirectly reflect the intracellular level of ROS and the degree of oxidative stress injury. Given that Ngb may act as an endogenous ROS scavenger, we evaluated the level of endogenous anti-oxidant agents, reduced glutathione (GSH), in which HCC cells were absent or present Ngb. The results showed that Ngb significantly increases the level of endogenous GSH, whereas Ngb knockdown reduces endogenous GSH level. In contrast, administration of exogenous anti-oxidant agents, GSH, the inhibition of Ngb was removed in HCC cells. In addition, we constructed Ngb mutants, which specifically inactivated the activity of ligand binding and guanine nucleotide dissociation inhibitor (GDI), according to previous structure and function investigation. These findings indicated that the activity of ligand binding is essential for the inhibition on HCC proliferation. However, the activity of guanine nucleotide dissociation inhibitor (GDI) may be not necessary for Ngb to suppress the proliferation of HCC. Futhermore, administration of exogenous GSH repeatly recovered the inhibition of Ngb on the proliferation of HCC, in which HCC cells the activity of ligand binding was inactivated. Our findings indicated that Ngb regulates the proliferation of HCC cells via coupling oxidative stress signal and the activity of ligand binding is responsible to the inhibition of Ngb.Accordingly, this study indicated that Ngb suppresses the proliferation of HCC cells via coupling oxidative stress signal and deregulating Raf-1/MAPK cascade. Ngb may act as an important role of endogenous ROS scavenger in HCC development. Given the important role of Raf-1/MAPK cascade in HCC development, we proposed that Ngb may be a therapeutic target for HCC treatment.