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Studies On The Expression And Functions Of Clorf61Gene In Human Hepatocellular Carcinoma

Posted on:2013-09-22Degree:DoctorType:Dissertation
Country:ChinaCandidate:H M HuFull Text:PDF
GTID:1314330395975889Subject:Microbiology
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Hepatocellular carcinoma (HCC) is ranked as the fifth most common human cancer, resulting in approximately600,000deaths every year worldwide. Hepatitis viral infection, alcoholism, and aflatoxin B1intake are the predominant causes of primary liver cancer. The main clinical treatments for HCC are resection and liver transplantation in non-cirrhotic and cirrhotic patients, respectively. However, the relatively asymptomatic initial phases of tumor progression and the high incidence of metastasis continue to be obstacles in the clinical treatment of HCC. Therefore, improving our understanding of the detailed mechanistic underpinnings of HCC tumorigenesis and metastasis is very important for the prevention and the treatment of HCC.Chromosome1open reading frame61(Clorf61) contains a468-bp open reading frame (ORF), which encodes a156-amino acid polypeptide of approximately17.2kDa. Clorf61was first reported to participate in the c-Fos signaling pathways that were involved in the cellular remodeling of the brain architecture. We have also previously reported that Clorf61is highly upregulated during weeks4to9of human embryogenesis, a critical period when most organs develop. Clorf61is implicated in embryogenesis during skeletal system development, including the bones of the vertebrae and limbs. A recent theory in tumor biology postulates that tumor formation and growth are maintained by populations of tumor-initiating cells (TICs) or cancer stem cells (CSCs) within the tumors. Previous studies have demonstrated that Clorf61is overexpressed in CD133+liver TICs isolated from the HCC cell lines Huh7and PLC8024. In contrast, the examination of a panel of human tissues revealed that Clorf61expression was undetectable in normal liver. Genomic amplification of chromosome1q22, the locus of Clorf61, is a common event in human HCC. However, the expression pattern of Clorf61in hepatocellular carcinoma (HCC) and its effect on HCC progression remain unclear.In this study, we detected the expression of Clorf61in different clinical samples including of normal liver, hepatitis, hepatic cirrhosis and HCC patients. Clorf61expression was associated with liver disease progression. Clorf61was upregulated in hepatic cirrhosis tissues and further upregulated in primary HCC tumors. Moreover, hepatitis B virus (HBV)-positive HCC patients exhibited significantly higher levels of Clorf61expression than HBV-negative HCC patients(P<0.05). In addition to the patient tissue samples, we also examined Clorf61levels in the HCC cell line HepG2(no HBV integration) and HepG2.2.15(with HBV integration). The results showed that HepG2.2.15expressed significantly higher levels of Clorf61protein than HepG2cells.The highly malignant HCC cell lines BEL7402and Huh7exhibited high expression levels of Clorf61protein, whereas the less malignant hepatoma cell line FHCC98exhibited moderate expression of Clorf61protein. Clorf61protein was mainly sub-cellular distributed in the cytoplasm.The ectopic expression of Clorf61in the non-malignant normal L02cells promoted cellular proliferation and colony formation in vitro. PI-staining and FACS analysis were performed to analyze alterations to the cell cycle. And the data indicated that overexpression of Clorf61resulted in an increase in the S-phase population with a concomitant decrease in the Gl population. Western blotting was used to analyze the expression of cell cycle-related proteins. The results indicated that the L02-GFP-Clorf61cells expressed higher levels of Cyclin El, Cyclin D1, Cyclin D3and CDK2than the L02-GFP cells. In addition, the L02-GFP-Clorf61cells exhibited significantly more wound closure than the L02-GFP cells after36hours in the wound-healing assay, indicating that Clorf61markedly enhanced horizontal migration. Next, we further examined cell migration/invasion by using a transwell assay chamber/invasion chamber. The results showed that the overexpression of Clorf61substantially enhanced cellular motility and cell invasion capacity, compared with L02-GFP cells (P<0.01).The epithelial-mesenchymal transition (EMT) is an important mechanism associated with cancer invasiveness and metastasis. The protein levels of epithelial cell markers E-cadherin, and occludin were downregulated in L02cells with overexpression of Clorf61. Correspondingly, L02cells with low expression of the Clorf61protein revealed low levels of N-cadherin, vimentin, and snail, which are mesenchymal cell markers. Consistent with the western blot results, downregulation of E-cadherin, and upregulation of vimentin were also observed by immune-fluorescence analysis in the L02-GFP-Clorf61cells. One of characteristics of cells undergoing EMT is an increase in cell spreading. Microscopic analyses revealed that the morphology of L02-GFP-Clorf61cells changed from tightly packed colonies and an uniform cobblestone morphology to the spindle-shaped, fibroblastic mesenchymal morphology. Moreover, isothiocyanate-conjugated phalloidin staining also revealed that the L-02-GFP-Clorf61cells formed lamellipodia, which was consistent with the cell morphology results.In conclusion, we demonstrated here that Clorf61is frequently upregulated in HCC and that its expression is also elevated in HBV-infected HCC patients. We also showed that Clorf61plays an important role in HCC tumorigenesis and metastasis by accelerating cellular proliferation and colony formation, enhancing cell motility and invasiveness, and by inducing EMT. Therefore, our findings demonstrated, for the first time, the roles of Clorf61in HCC tumorigenesis and metastasis.
Keywords/Search Tags:Clorf61gene, hepatocellular carcinoma, epithelial-Mesenchymaltransition
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