Hepatocellular carcinoma (HCC), which constitutes 75%-80% of primary liver cancer, is one of the most common malignancies worldwide. Hepatocarcinogenesis is a complicated and slow process accumulating multiple genetic and epigenetic alterations. In spite of its prolonged pre-malignant stage, HCC is usually diagnosed late and of high aggressiveness. A better understanding of the genetic and epigenetic changes during HCC development and progression is of great importance to early diagnosis and treatment of HCC. Gain of chromosome 1q21 is one of most frequent genetic alteration in HCC and chromodomain helicase DNA binding protein 1-like (CHD1L) was recently identified to be responsible for this amplification. As a family member of SNF-2 like transcription factors, CHD1L plays an important role in HCC development via regulation of various downstream targets. In this study, a novel oncogene, sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 (SPOCK1), was identified as a CHD1L target. CHD1L protein directly bound to the promoter region (nt -1662 to +34) of SPOCK1 and activated its transcription. Clinically, overexpression of SPOCK1 was detected in 60% of human HCC samples and was significantly associated with advanced clinical stage (P=0.020), shorter overall survival (P=0.011) and poorer disease-free survival of patients (P=0.039). Functionally, the ectopic expression of SPOCK1 in HCC cells conferred strong tumorigenic ability, while shRNA-mediated SPOCK1 silencing abolished this effect. Further study showed that the SPOCK1-enhanced cell survival could be attributed to its anti-apoptotic effects. SPOCK1 could suppress HCC cell apoptosis through the activation of AKT and subsequent inhibition of the (cytochrome c)-(caspase-9)-(caspase-3) pathway. In addition to its tumorigenic roles, the overexpression of SPOCK1 in HCC cells conferred strong metastatic ability via MMP9-mediated extracellular matrix remodeling. In addition to genetic alterations, epigenetic changes also get increasing attentions due to their profound effects on gene activity and expression. A-to-I RNA editing is a post-transcriptional epigenetic modification which converts a site-selective adenosine nucleotide into inosine. The importance of RNA editing has long been underestimated because most of RNA editing modifications occur in a subtle way. The next-generation sequencing provides enough depth to unravel this mystery. The transcriptome sequencing data obtained from this study identified an A-to-I RNA editing at codon 367 (Ser→Gly) of antizyme inhibitor 1 (AZIN1). A high modification rate of AZIN1 was found to be prevalent in HCC specimens and closely associated with HCC pathogenesis. Adenosine deaminase acting on RNA-1 (ADAR1), but not ADAR2 or ADAR3, was responsible for AZIN1 RNA editing. This recoding editing event conferred “gain-of-function” phenotypes as manifested by augmented tumorigenic capabilities and higher aggressive potentials. Compared with wild-type AZIN1 protein, the edited form possessed stronger affinity to antizyme. As a result, edited AZIN1 demonstrated higher protein stability and ensuing neutralization of the antizyme-mediated degradation of ODC and CCND1 oncoproteins. The rescued ODC and CCND1 robustly accelerated cell proliferation thereby promoting HCC development. In conclusion, two novel molecular mechanisms, (CHD1L)-(SPOCK1)-(AKT) and (ADAR1)-(edited AZIN1)-(ODC/CCND1), were delineated during HCC initiation and progression. Also, a causal link between RNA hyper-editing activity and cancer development was established for the first time in this study. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. The development and progression of HCC is a multistep process which attributed to the accumulation of genetic alterations. Recently, mounting evidence has suggested the role of non-coding RNAs (ncRNAs) as the master driver of carcinogenesis, through their regulation on different oncogenes and tumor suppressive genes. Deregulation of ncRNAs was frequently observed in multiple types of cancers, including HCC. Herein, we demonstrated the aberrant expression pattern of miRNAs and lncRNAs in human HCC and investigated their functional roles in promoting hepatocarcinogenesis. Deregulation of miRNAs was previously demonstrated as a common event in human HCC, and miR-142-3p and miR-142-5pwereidentified as one of the significantly down-regulated miRNAs in HCC. Down-regulation of miR-142-3p and miR-142-5p was frequently observed in HCC patients and their expressions were progressively decreased along the multi-steps HCC development and progression. Functionally, overexpression of miR-142 has significantly inhibited HCC cell migration and invasion. Ectopic expression of miR-142 also markedly attenuated stress fiber formation and disrupted the cytoskeleton organization of HCC cells. Mature miR-142-3p and miR-142-5p, which derived from the same miRNA precursor were shown to collaboratively inhibited HCC cell migration through targeting different components of the key pathways regulating cell motility. On the other hand, we demonstrated the aberrant expression of lncRNAs in HCC by profiling of 88 well-annotated lncRNAs in 20 pairs of primary HCC and their corresponding non-tumorous liver. HOXA distal transcript antisense RNA (HOTTIP)was identified as the most frequently up-regulated lncRNA in HCC. Functionally, knock down of HOTTIP significantly attenuated cell proliferation in HCC cells and markedly abrogated tumorigenicity in nude mice. Knockdown of HOTTIP had lead to a global reduction in the HOXA genes expression, which are highly expressed in human HCC. Our data suggested that HOTTIP may regulate the expression of its neighboring protein-coding genes and contribute to the development of HCC. We also investigated the up-stream regulation of HOTTIP and identified miR-125b and miR-29a as regulators of HOTTIP in HCC. Clinically, miR-125b and miR-29a exhibited a reverse expression pattern to HOTTIP in HCC. Ectopic expression of miR-125b and miR-29a abolished HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Intriguingly, we also identified a negative feedback relationship between HOTTIP and miR-125b. Taken together, our findings suggested the up-regulation of HOTTIP may be attributed to the down-regulation of miR-125b and miR-29a in HCC, and the sophisticated regulatory network between HOTTIP and miR-125b has further increased the complexity of gene regulation in HCC. In conclusion, we demonstrated the dysregulated expression pattern of miRNAs and lncRNAs in HCC and well illustrated their functional roles in promoting hepatocarcinogenesis. From the studies of miR-142 and lncRNA HOTTIP we appreciated the complex interactions and regulations between different ncRNAs. Taken together, our study has enriched the current knowledge on ncRNAs and their involvements in HCC development. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Liver fibrosis is an important reversible stage in progress of most chronic liver diseases (CLDs). The excess hepatic wound healing response against chronic liver injury results in extracellular matrix proteins accumulation and fibrosis. Oxidative stress, liver inflammation and/or hepatic steatosis contribute to this process. Until now, little is known how dietary lipids can influence liver’s pathophysiology. The effects of lipids on CLD progression may depend on their amount and the quality of fatty acids as well as the degrees of saturation. The investigation of liver fibrosis will help to understand the pathogenesis of CLDs and develop potential nutritional therapeutic approaches. The specific aim of this study was to investigate the effects of different high fats consumption in liver fibrosis by feeding the normal and carbon tetrachloride (CCl4)-treated animals with the diets enriched with following oils: corn oil rich in ω-6 polyunsaturated fatty acids (PUFAs), extra virgin olive oil (EVOO) high in ω-9 monounsaturated fatty acids (MUFAs), and lard enriched with saturated fatty acids (SFAs) for 4 weeks. The differentially expressed liver proteins in this process were identified by two-dimensional gel electrophoresis based proteomics to explore the molecular mechanisms. The proteomic analysis revealed characteristic differences between (i) normal and fibrotic livers (Chapter 3), and between the fibrotic livers treated with (ii) low fat versus high fat (20% w/w corn oil, Chapter 4) and among the high fats, between the diet enriched with corn oil versus (iii) EVOO (Chapter 5) and lard (Chapter 6). Among the identified proteins, collagen synthesis related protein prolyl 4-hydroxylase, oxidative stress related protein alpha-1-antitrypsin, free radical scavenger Cu/Zn superoxide dismutase and Calcium homeostasis regulator calreticulin and regucalcin were found to involve in CCl4-induced liver fibrosis. The results show that corn oil enhanced the hepatic steatosis but had no significant effects on fibrogenesis; the expression of several stress proteins like heat shock protein 75 kDa, and lipid metabolism related protein enoyl-CoA hydratase domain-containing protein 3 were found increased in high corn oil consumption animals with CCl4-treatment. Histological evaluations showed that olive oil could attenuate, and lard oil aggravate the liver damage induced by CCl4. Compared to corn oil, high EVOO diet rich in MUFAs decreased the lipid peroxidation and collagen accumulation in liver. Several protein related to antioxidant effects, including peroxiredoxin-1, thiosulfate sulfurtransferase and thioredoxin domain-containing protein 12 were found have higher expression level in high EVOO intake animals. In contrast, lard rich in SFAs intake leaded to macrovesicular steatosis and advanced fibrosis, and decreased the expression of antioxidant related glutathione S-transferases. Interestingly, S-adenosylmethionine synthesis related enzyme methionine adenosyltransferase was found up-regulated in lard intake animals, suggests the modification of DNA methylation was implicated in lard fed animals, while the demethylation on the promoter of profibrogenic gene was found, confirmed the lard consumption has the epigenetic modification effects in liver injury. Together, these findings give further insight into the pathobiology of CLDs. The data also helped to address the issue that different degrees of saturation of dietary lipids may affect liver fibrosis with different mechanistic actions. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) accounts for 70-85% of liver cancer, which is the sixth most common cancer in the world. Prognosis of HCC is dismal with little chance of complete recovery after diagnosis. It is of essence to discover the key molecules involved in the tumor progression. This could help earlier detection of HCC and establish targeted molecular therapies. Cyclin E1 (CCNE1) is a cyclin molecule responsible for the transition from G1 to S phase of the cell cycle and is often dysregulated in human cancers. CCNE1 is reported with overexpression in about 30-70% of HCC cases. It expresses in tumor cells as a ladder of proteins and as low molecular weight CCNE1. The study is aimed to investigate the role of CCNE1 in HCC. From the local cohort of HCC patients, 6 out of 13 patients (46.2%) of HCC tumor tissues were found with CCNE1 overexpression compared with the non-tumor tissues by western blotting. The presence of three CCNE1 isoforms in HCC was detected. The expression of total CCNE1 and each isoform varied independently among the studied HCC cell lines, with HepG2 having the highest expression and 97L the lowest. To extend our study on the regulation of CCNE1 expression, the expression of selected four genes associating with the CCNE1 expression and functions was studied by quantitative PCR (qPCR). F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase (FBXW7) and cullin 3 (CUL3), the two genes responsible for CCNE1 degradation, had increased expression in the HCC cell lines with higher CCNE1 expression. Cyclin A (CCNA2), the downstream cyclin molecule of CCNE1, also had higher expression in these cell lines. In contrast, the expression of cyclin dependent kinase 2 (CDK2), the catalytic partner of CCNE1, had the least difference among the six HCC cell lines compared to other three genes. To characterize the role of CCNE1 isoforms in HCC, CCNE1 isoform 1, 2, and 3 were overexpressed in PLC cells and such overexpression remained even after 8 passages in culture. In flow cytometric analysis, GFP signal in cell culture population was viewed to observe the transduction efficiency. The vector control showed the strongest GFP signal, followed by CCNE1 isoform 3 showing dim signal. CCNE1 isoform 1 and 2 almost showed no signal. In the functional studies, the overexpression of CCNE1 isoform 3 could increase proliferation and migration of HCC cells. In summary, CCNE1 could promote proliferation and migration of HCC cells through elevated expression of CCNE1 isoform 3. / published_or_final_version / Surgery / Master / Master of Philosophy

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second leading cause of cancer-related deaths in the world. It is a disease with poor prognosis with unsatisfactory long-term survival of patients, and thus new strategies to control this disease are warranted. T helper (Th) 17 cells and IL-17 have recently been detected with increased frequency in a number of tumors including HCC. Its role in tumor remains controversial but its presence in HCC has been linked to disease progression, possibly involving angiogenesis. Th17 cells could be homed to inflammatory sites such as tumor microenvironment via CCR6/CCL20 axis and expand locally, and studies from other inflammatory diseases such as autoimmune disease has shown that the gut is the potential source of Th17, where its induction is affected by signals from gut microbiota. Yet this link is not yet shown in extra-intestinal tumors. Probiotics are living microorganisms, which when administered in adequate amounts confer a health benefit on the host. They have been reported to relieve chronic inflammatory diseases in animal and in human intervention studies. It is believed that probiotics regulate signals to gut antigen-presenting cells, which act as the pivot in modulating the systemic immune responses and inactivated bacteria also exhibited immunomodulatory effects in this regard. Accordingly, it was hypothesized that oral feeding of probiotics to HCCbearing animals may affect Th17 polarization and distribution and thereby modulate tumor microenvironment, which may have beneficial effect in tumor development, possibly via affecting angiogenesis. To address this hypothesis, wild-type C57BL/6 mice were fed with different heat-inactivated or viable probiotics– Lactobacillus rhamnosus GG (LGG), Escherichia coli Nissle 1917 (EcN), VSL#3 or mixture of probiotics − Prohep (heat-inactivated LGG, heatinactivated VSL#3 and viable EcN) either one week in advance or at the time of subcutaneous tumor inoculation. Probiotic feeding had improved survival in tumor-bearing mice, slowed down tumor growth and reduced tumor burden when monitored for 38 days. Probiotics showed better efficacy when feeding was given in advance. The anti-tumor effect was related to reduced angiogenesis and reduced IL-17 serum and gene expression within tumor. The mechanistic link between IL-17 modulation and tumor development was further studied in animals by IL-17 neutralization. The anti-tumor efficacy of probiotics, in relation to tumor growth and angiogenesis, was lost after IL-17 neutralization, which was linked to recruitment of myeloid suppressor cells. Since cells from both adaptive and innate immune systems could secrete IL-17, the source of IL-17 production was then identified, and found that Th17 was the major IL-17 secretor being modulated by probiotic feeding. Reduced homing of Th17 to tumor via circulation, with a tendency being recruited from gut was observed. Probiotics-mediated Th17 cell modulation in the gut by inducing the skewing of IL-10 secreting type1 regulatory T cells via dendritic cells may link to limited IL-17 mediated angiogenesis in the tumor microenvironment. With better understanding of the immunomodulation properties of probiotics, prophylactic or therapeutic efficacy in management of other inflammation-associated cancer can be availed. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

abstract / Surgery / Doctoral / Doctor of Philosophy

The primary goal was to study the influence of adipocyte number and volume, inflammation, insulin resistance, and genetic factors on indicators of liver injury, surrogate marker of non alcoholic fatty liver disease (NAFLD). The secondary goal was to explore the occurrence of NAFLD and its relationship with variations in liver function biomarkers. The first objective was to determine the association of plasma levels of monocyte chemoattractant protein-1 (MCP-1) with omental adipocyte number, insulin resistance and circulating concentrations of liver injury markers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in unrelated baboons. Significant associations of MCP-1 with other measured traits were established. The second objective was to examine if adiposity-related parameters are under genetic influence and to evaluate their genetic correlations with AST in pedigreed baboons. Adipocyte volume and number, body weight and plasma AST were heritable. Genetic correlations between adiposity-related phenotypes and AST were significant. A genome wide scan yielded a strong signal for adipocyte volume on chromosome 6. The third aim was to explore the genetic factors that influence variations in plasma levels of [gamma] glutamyl transferase (GGT) and albumin (ALB), and to evaluate their genetic correlations with cardiovascular risk factors in pedigreed baboons. Significant linkages for GGT and albumin were identified on chromosome 20_22 and chromosome 10, respectively. Genetic correlations between ALB and cardiovascular risk factors were significant. No statistically significant associations were found between GGT and cardiovascular-related phenotypes. The fourth objective was to investigate the prevalence of NAFLD and its association with altered liver protein levels in unrelated baboons. The influence of weight and insulin resistance on the occurrence of NAFLD was inconclusive. Significant relationships between the variations in plasma levels of liver injury biomarkers and severity of the disease could not be established. In conclusion, the first three studies provided observational and genetic evidence of a relationship between liver function markers and adiposity-related factors in baboons. However, the results of the fourth study do not provide conclusive evidence to suggest that body weight and insulin resistance play a significant role in the development of NAFLD in these baboons.

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences