The Study On Metabolic Abnormality And N-glycosylation In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death in the world. China is a high level epidemic region of HBV infection.The relative risk for hepatocellular carcinoma(HCC) was 200 among patients with the positive of HBsAg compared people with negative of HBsAg.At present, the risk factors for liver cancer derived from large scale epidemiological studies revealed that the metabolic syndrome (MS) are closely related with liver cancer, and even could be the independent risk factors for the occurrence and progression of liver cancer. Surgery remains the best way for a curative therapy of HCC.However, tumor recurrence and metastasis is the main obstacles to improve prognosis.The key of the treatment of HCC is principle of early detection, early diagnosis and early treatment .So,preventing the recurrence and metastasis after surgery is a very important step to improve the prognosis of HCC.And predicting and founding the recurrence and metastasis of tumor earlier seems more important.practice.The project is composed of two parts, which described the influence of occurrence and development of HBV-related HCC respectively.PartⅠ: Association between metabolic abnormalities and HBV related hepatocellular carcinoma in Chinese: A cross-sectional studyBackground: Previous studies suggested that the abnormality of metabolism is a newly identified risk factor in HBV-related hepatocellular carcinoma (HCC). The association between metabolic factors and hepatocellular carcinoma (HCC) has not been clarified up to now. This study was conducted to investigate the prevalence of metabolic abnormalities in HCC and to probe the association between metabolic parameters and liver function as well, so as to evaluate the interactions between metabolism and the development of HBV-related HCC.Methods: Totally 179 cases of HBV-related HCC, who were surgically treated and pathologically confirmed were enrolled. HBV carrier (n=100) and healthy control (n=150) were recruited from routine physical examination during the same period. Body mass index (BMI) was obtained from medical documentation. All the metabolic-related parameters and liver function tests were determined with routine biochemical or immunological analytic methods. Malondialdehyde (MDA) and total antioxidant capacity(TAOC)were detected by chemical analytic methods. A stratified analysis was conducted according to BMI, glycated albumin (GA), free fatty acids (FFA), and the relationships between the metabolic-related parameters and liver functions were analyzed in HCC and control subjects.Results: HCC group showed significantly high levels of mean BMI, serum glucose, low serum lipids levels than controls (P<0.05). Acquired by stratified analysis, the higher the BMI, the higher level of insulin and homeostasis model assessment for insulin resistance (HOMA–IR) (P<0.01) were found in HCC. Elevated level of MDA andγ-glutamyltransferase (GGT) were revealed in those with high serum FFA level for the first time. Strong associations between metabolic factors and liver function were shown in HCC (P<0.05). Higher GA level was strongly associated with increased risk of cancer compared to healthy controls (OR=9.87, 95% confidence interval: 1.86~52.29). Serum triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels were negative contributory factors for HCC (OR=0.05, 95% confidence interval, 0.01~0.27 and OR= 0.32, 95% confidence interval, 0.11~0.95, respectively).Conclusions: Metabolic abnormalities are closely associated with the occurrence and development of HBV-related HCC. Oxidative stress and/or lipid peroxidation might be involved in the pathogenesis and accelerate liver function impairments in HCC. PartⅡ: Biological function and significance of N-acetylglucosaminyltransferase III and N-acetylglucosaminyltransferase V in hepatocellular carcinomaObjective: By constructing a recombinant eukaryotic expression vector containing the functional region of human GNT-III and GNT-V gene and transfecting this vector into human hepatoma carcinoma cell line to obtain transient-expression cell line,a lentiviral vector-mediated RNA interference (RNAi) of GNT-V was constructed, stable transfer cell lines were acquired. we meant to investigate the impact of the expression of gene on the proliferation,adhesion and invasion of hepatoma carcinoma cells. In order to investigate the functions of GNT-III and GNT-V in HCC,We have detected their expressional characteristics and correlations with clinical characteristics in human HCC and surrouding liver tissue, which settled the foundation for the further study of the effect of invasion and metastasis of human hepatocellular carcinoma(HCC).Methods: GNT-III and GNT-V were amplified using reverse transcription polymerase chain reaction(RT-PCR).Then the gene was cloned to PCMV-GNT-III and pEGFP-N1-GNT-V ,then Liposome was used to transiently transfer GNT-III and GNT-V into human hepatoma carcinoma cell lines,After selecting effective sequence of GNT-V RNAi, and then packaging plasmids of Lentiviruse and transfected to cell lines successfully and the result of expression was detected by RT-PCR, The effect of GNT-III and GNT-V on the proliferation of cells was detected by CCK8 assay and their adhesive abilities to matrigel was also detected by MTT assay. The effects of GNT-V on invasion of the transfected cells were investigated by reconstituted matrigel invasion experiment.The effects of GNT-V on migration of the transfected cells were investigated by wound closure assay. Expression of GNT-III and GNT-V in cancerous and adjacent non-cancerous tissues from 36 HCC patients was detected by Realtime PCR. Expression activity of GNT-III and GNT-V was detected by Lectin blot.Results: The recombinant eukaryotic expression vector pCMV-GNT-III and pEGFP-N1-GNT-V were obtained and they were confirmed that GNT-III and GNT-V cDNA was inserted into the eukaryotic expression vector correctly using PCR, digestion identification and sequencing.It was showed that GNT-III and GNT-V gene had been transfected into human hepatoma carcinoma cell lines using liposome and the result was detected by RT-PCR. The proliferation of cells overexpressing GNT-V was enhanced, while compared with cells of control groups. The overexpression of GNT-V obviously increased the adhesion of cells to matrigel (P<0.05),when compared with cells of other groups. The invasion of the cells expressing GNT-V gene was significantly advanced in Matrigel-coated invasion chambers(P<0.01),when compared to cells of control. Enhanced migration of GNT-V transfected cells was revealed by wound closure assay. There was no significant changes of biological effects in the group transfected with lentivirus vector Expression of GNT-III and GNT-V mRNA in cancerous tissue was significantly lower than that of its paired adjacent non-cancerous tissue. The affinity glycosylation structure of PHA-E and PHA-L were significantly higher in the adjacent tissues than cancer tissues; tissue.There was no significantly correlation between the expression of GNT-III or GNT-V and clinical and pathological factors .Although the higher expression of GNT-V showed a higher recurrence and decreased disease-free survival, which was no significant difference (P>0.05).Conclusion: The recombinant eukaryotic expression vector pCMV-GNT-III and pEGFP-N1-GNT-V and the transient expression GNT-III and GNT-V cells were successfully obtained,which settled the foundation for the father study of founding out the impact on proliferation, adhesion and invasion of GNT-III and GNT-V in hepatoma carcinoma cells through vitro experiment. It was showed that the abilities of adhesion to Matrigel, proliferation and migration of hepatoma carcinoma cells transfected by GNT-V could be promoted through vitro experiment; Expression of GNT-III and GNT-V mRNA in cancerous tissue was significantly lower than that of its paired adjacent non-cancerous tissue.