Preliminary Study On The Biological Function Of VASN Protein And Its Regulatory Mechanism

Primary carcinoma of the liver(PCL) is one of the most common cancer in clinical. It is a great threat to the human health in modern society due to the high mortality. Especially in China, the incidence and mortality rates of liver cancer are among the front ranks of the world, and showed a rising trend. Primary carcinoma of the liver have a variety of types, hepatocellular carcinom, hepatoblastoma, cholangiocarcinoma, sarcoma, but primary type is hepatocellular carcinoma and accounts for more than 90 %.Early diagnosis and treatment of liver cancer is critical to the prognosis and survival time. Aalpha fetoprotein(AFP) was discovered in 1965 and reported as the first liver-specific tumor markers with diagnostic value, but also the only clinical diagnosis markers of primary liver cancer. However, a large number of clinical data has found that some cirrhosis patients detected with long-term positive AFP, meanwhile there is still about 30% liver cancer patients tested AFP negative performance in serum. In recent years, a number of hepotacellular carcinoma related signaling pathways have been clearly explained, however, the condition of difficult early diagnosis and poor prognosis remains unchanged. Therefore, it is still imminent to take a more in-depth understanding of the molecular mechanism of liver cancer and find a new serum markers or diagnostic and therapeutic targets.In our previous study, we used a substractive EMSA-SELEX technology to screen an AFP negative serum marker from patients with extrahepatic metastasis of HCC. We found a candidate marker molecules-VASN(Vasorin, slit like 2) protein and confirmed by experiments. Results showed that the expression levels of VASN is higher in HCC serum, tissues and cells than in normal liver serum, tissues and cells, and high expression of VASN promote cell proliferation and migration significantly. The main contents of this paper is to further explore biological function and regulation mechanism of VASN protein in hepatocellular carcinoma, and topic is divided into two parts:The first part: Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells.Vasorin(VASN) is a type I transmembrane protein that plays important roles in tumor development and vasculogenesis. In this paper, we showed that VASN could be a key mediator of communication between tumor cells and endothelial cells. We confirmed for the first time that Hep G2-derived VASN can be transferred to human umbilical vein endothelial cells(HUVECs) via receptor mediated endocytosis of exosomes, at least in part through heparin sulphate proteoglycans(HSPGs). The Hep G2-derived VASN containing exosomes promote migration of recipient HUVEC cells. Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.We first explored the function of VASN in vascular endothelial cells. The bioimformation analysis revealed VASN is mainly expressed in cell membrane and secreted. We examined the culture supernatant of Hep G2 cells and detected in the culture supernatant containing a large amount of secretory VASN. After HUVECs were co-cultured with Hep G2 culture supernatant and found that VASN protein expression increased in co-cultured HUVECs, while m RNA expression of VASN were not changed, suggesting that rising VASN protein level in HUVECs may come from external sources such as liver cancer cells.Cancer cells may communicate with endothelial cells by secreting free vascular endothelial growth factors such as VEGF, or by releasing membrane vesicles such as exosomes to transfer functional molecules including oncoproteins into recipient cells. We examine VASN expression in supernatant and exosomes. The results showed that VASN can be detected both in the culture supernatant and exosomes and can be detected VASN, and VASN levels in the culture supernatant after exosomes removed are significantly reduced. These results demonstrate VASN can be present in exosomes.To further explore the biological function of exosomal VASN, we isolated Hep G2-derived exosomes and incubated them with HUVECs for 24 hr. Result showed that the protein levels of VASN in whole cell extracts of HUVECs were increased, suggesting exosomal tranport might be a communication between cancer cells and endothelial cells. Pre-silencing VASN expression in Hep G2 with si RNA could block the VASN elevation in Hep G2-derived exosomes treated HUVECs may be because of lower VASN in exosomes and the transfer of VASN into HUVECs by Hep G2-derived exosomes showeda dose-dependent manner. The exogenous VASN with myc-tag was transiently expressed in Hep G2 cells, and the internalization of exosomal myc-VASN into HUVECs was visualized by immunofluorescence with anti-myc antibody and fluorescence labeled secondary antibody. All the above implies an exosomes specific intercellular transfer of VASN from Hep G2 to HUVECs.Cells appear to take up exosomes by a variety of mechanisms including endocytosis, macropinocytosis, phagocytosis, and lipid raft_mediated internalization. Among which, the heparin sulphate(HS) proteoglycans(HSPGs) are recently reported to play an important role in the cell surface adsorption and internalization of exosomes. We use heparin(HS mimetic) as a competitive inhibitor of cell surface receptors dependent on HSPGs coreceptors to block the HSPGs pathway. After culture the Hep G2-derived exosomes with HUVECs in presence of heparin, the increased VASN in recipient cells were decreased in a heparin dose-dependent manner. This implies HSPGs involves in the transfer of exosomes VASN into HUVECs. And we performed the same exosome up-take experiment in different repient cells(L02, He La, U937) demonstrated the transfer of VASN from Hep G2 via exosomes is cell type specific in HUVECs. Corresponding with HSPGs expression in these cell lines.To analyze the biological effects of intercellular transfer of VASN from Hep G2 to HUVECs, we evaluated the proliferation and migration of HUVECs treated with Hep G2 supernatant and Hep G2-derived exosomes. Neither Hep G2 supernatant nor Hep G2-derived exosomes significantly affected the proliferation of HUVECs. However, significant effects were observed on HUVECs migration, indicating that VASN transported by Hep G2-derived exosomes can effectively accelerate HUVECs migration.In conclusion, we identified a novel pathway regulating tumor metastasis via the transmembrane protein VASN. We believe that VASN-positive exosomes provide a metastatic signal from primary tumor cells to the surrounding cells. Exosomes act as a carrier of this signal by mediating cell-cell communication in the progression of cancer, such as hepatocellular carcinoma, and exosomes-mediated transfer of metastatic factors could be a means for cells to induce cell migration in paratumor tissues. In the present study, we discovered that a functional protein highly expressed in a transformed cell could affect the biological fate of an endothelial cell via exosomes-mediated transfer Detailed investigation of this mechanism could help elucidate a novel pathway in the progression of tumor metastasis.The second part: mi R-152 reduces human umbilical vein endothelial cell proliferation and migration by targeting ADAM17.The disintegrin–metalloproteinases(ADAMs) family is a group of cell-surface glycoproteins. ADAM17 is one of the ADAM family, major function of ADAM17 is used as a metalloproteinase to activate tumor necrosis factor-α secretion according to reports. Meanwhile ADAM17 also regulate VASN secretion in the same way. Report found ADAM17 could cut the extracellular domain of membrane-type VASN into secretion-type VASN. After the extracellular domain shear off and secreted to blood circulation as a secreted VASN exert biological functions. In recent years, an increasing number of reports was found to have a close link between tumor metastasis and invasion and mi RNAs, mi RNAs regulating cell adhesion, matrix degradation, epithelial mesenchymal transformation(EMT) and angiogenesis related genes play a role in the regulation of tumor development. Therefore, we investigated the mi RNAs regulatory mechanisms of ADAM17.Bioinformatics predicted that ADAM17 may be a target of mi R-152 in human cells. We constructed a recombinant luciferase reporter plasmid containing mi R-152 target sites in ADAM17 3’-UTR and conducted a dual luciferase reporter gene testing. Our result found mi R-152 mmics can bind to mi R-152 target sites in ADAM17 3’-UTR and inhibit luciferase activity of the reporter gene, but has no effect on the mutant control plasmid. Over expression of mi R-152 incurred down regulation of ADAM17 m RNA levels and protein levels. Under hypoxic conditions, mi R-152 was low expressed due to the effects of low oxygen environment, caused increased expression of ADAM17, which led to an increase in TNF-α shedding. ADAM17 expression which silenced with si RNA also inhibits invasion and migration of HCC cells, and in accord with the effect of mi R-152 overexpression. Combine with all these evidences that we confirm the level of mi R-152 were negatively regulate the transcription of ADAM17 and involved in migration and invasion of HCC.