published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Clinical Oncology / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

Vitamin A is known to have an important role in cellular differentiation and proliferation. In addition to regulating normal cellular processes vitamin A has also been shown to possess potent antineoplastic activity. The work in this dissertation characterizes the role of retinoic acid (RA) in cellular transformation and tumorigenesis with known oncogene involvement. These studies were initiated by examining the effects of RA on human carcinoma cell lines which express an activated c-ras gene. The bladder carcinoma, EJ/T24 (c-rasᴴ) and the two lung carcinoma cell lines, LXl (c-rasᴷ) and A2182 (c-rasᴷ), were not sensitive to RA. No inhibition of anchorage- or density-dependent growth was observed. Therefore, since these in vitro markers of transformation indicated a lack of effectiveness of RA on carcinomas containing a c-ras gene, retrovirally transformed cells were tested for RA sensitivity. Kirsten murine sarcoma, Balb/c murine sarcoma virus, and Simian sarcoma virus transformed NIH/3T3 and NRK cells were used in these studies. In contrast to the human carcinoma cell lines, anchorage-independent growth of some of the virally transformed cells was very sensitive to inhibition by RA. Anchorage-independent growth of KNRK and SSVNRK cells was sensitive to high concentrations (5 μM) of RA; whereas, Balb/cMSV3T3 and SSV3T3 were sensitive to 1-20 nM RA. BALB/cMSVNRK anchorage-independent growth was stimulated 3.5 fold by 1 μM RA. KNRK displayed a 60% reduction in anchorage-dependent growth at 10 μM RA while little inhibition was observed with the other retrovirally transformed cells. A high level of sensitivity to RA inhibition of anchorage-independent growth was correlated with the presence of cytoplasmic retinoic acid binding protein (CRABP). This indicated that CRABP may have some role in the inhibition of retrovirally induced cellular transformation. RA was shown to significantly reduce the incidence and size of Balb/cMSV3T3 cell tumors in nude mice. The inhibition of tumorigenesis in vivo therefore confirmed the results observed in vitro. To investigate the mechanism by which RA was acting to inhibit retroviral transformation, v-onc mRNA levels were examined. RA had no effect on v-onc mRNA levels in cell lines sensitive to the inhibition of transformation. The effect of RA on the relative rate of synthesis of p21, the transforming protein of KMSV and Balb/cMSV, was investigated. No effect of RA was observed in any of the cell lines. Also, GDP binding by p21 in KNRK cell was unchanged by RA treatment indicating that the functional activity of this transforming protein was not modified. RA does appear to be effective in inhibiting retrovirally induced cellular transformation and tumorigenesis. Evidence presented here indicates that this inhibition is not due to a direct effect of RA on the expression of the v-onc gene and/or gene product. Therefore, some other essential cooperating event(s) occurring within the cell are being acted upon by RA.

MSc. (Biochemistry) / GLOBOCAN 2008 Survey reported that 12.7 million cancer cases with 7.6 million cancer deaths occurred with an astonishing 56% of these cases and 64% of these deaths allocated to economically developing countries, such as South Africa. Statistics are alarming concerning cutaneous malignant melanomas (CMM) with the World Health Organisation (WHO) estimating that 132 000 new cases of CMM arise per annum internationally with CMM incidence rates showing an increase of 28% in men and 12% increase in women worldwide; whilst the Cancer Association of South Africa (CANSA) has reported that skin cancer is the most common cancer in South Africa, with an estimated 20 000 new cases being reported per annum. Normal cells progressively transform into malignant tumours by a process that requires sequential acquisition of mutations in a genome damaged by various intrinsic and exogenous incidences resulting in two distinct and functional outcomes: 1) activation and/or expression of unfavourable oncogenes and 2) inactivation of tumour suppressor genes that code for proteins involved in checkpoints to cell proliferation or cell death. Transformation of dendritic melanocytic epidermal skin cells give rise to different types of skin cancers with CMM being predominant with poor prognosis and 90% of all deaths associated with cutaneous type tumours and CMM has been classified as a multifactorial disease where both environmental and genetic factors/mutations interact in concert to contribute to CMM susceptibility. Conceptual progress in the last decade has added two emerging hallmarks showing increased potential in generality to the already six known hallmarks of cancer, namely reprogramming of cellular energy metabolism and evasion of immune destruction by T and B lymphocytes and macrophages, enabled by core hallmark cancer characteristics such as genome instability and tumour-promoting inflammation. The Warburg Effect has been described, in terms of metabolic particularities of cancers, as an increased glucose uptake, via a shift in energy production from oxidative phosphorylation to a glycolytic pathway, with increased extracellular lactate release by tumours resulting in a consequent decrease in pH in the surrounding tissues, even in the presence of oxygen. This effect contributes to proliferation, invasiveness, metastasis and angiogenesis of malignant cells. Thus, chronic and uncontrolled cell proliferation, representing the essence of tumour growth, involves not only deregulatory control of cell proliferation but also a parallel adjustment to energy metabolic pathways in order to fuel cell growth and division. Over the last twenty years, studies have shown that the concept of programmed cell death (PCD), by apoptosis, serves as a natural barrier to cancer development where both the intrinsic and extrinsic apoptotic circuits conclude in the implementation of progressively...

University of Macau / Institute of Chinese Medical Sciences

運用miRNA微陣列手段，我們篩選到一批在結直腸腫瘤內高表達和低表達的miRNA。其中miR-133a是在腫瘤中最顯著降低的miRNA之一， 但其在腫瘤的發生發展過程中的作用尚未可知，因此我們選擇miR-133a最為研究目標，論證其的生物學功能，分子機理，及其在結直腸癌中的靶分子。 / 我們首先在較大規模樣本中驗證miR-133a的低表達。定量PCR結果顯示，對比正常的癌旁組織，miR-133a在94例結直腸癌組織中顯著低表達（p < 0.001）。我們進一步分析miR-133a在9種常用的腫瘤細胞系內的表達情況。對比正常的直腸組織，miR-133a在9種常用的結直腸細胞系內的表達均明顯下降。在腫瘤發生發展過程中，癌細胞趨向於降低具有抑癌功能的基因的表達。因此我們推測miR-133a是一個潛在的腫瘤抑制miRNA。 / 為了論證我們的假設，我們首先選取miR-133a低表達的結直腸癌細胞系HCT116和LoVo最為研究模型，將miR-133a在這兩種細胞系內過表達。升高的miR-133a可以抑制腫瘤細胞生長（p < 0.01和p < 0.05），抑制腫瘤克隆集落形成（p < 0.01）。我們進一步發現過表達miR-133a可以抑制裸鼠體內腫瘤的生長（p < 0.05）。細胞週期分析顯示miR-133a抑制細胞生長的能力表現為誘導腫瘤細胞週期阻滯於G0/G1期。細胞週期特異性CDK抑制蛋白p21和p27對於細胞週期調節十分關鍵。基於此項觀察，我們進一步分析了p21和p27的表達。Western-blot實驗證實過表達miR-133a可以促進HCT116和LoVo細胞內p21和p27的蛋白上調。但miR-133a在p53突變型的HT29過表達後並沒有引起p21的變化。通過對比p53野生型和突變性細胞系，我們發現p53對於miR-133a誘導p21是十分關鍵的。為了證明miR-133a可以引起p53的活性，我們通過啟動子螢光報告實驗證實，miR-133a不僅可以促進p53結合DNA 的能力，而且可以引起p21啟動子的活性。其次，我們發現在p53野生型細胞株HCT116中，轉染si-p53可以拮抗miR-133a誘導p21。我們通過蛋白降解實驗發現，miR-133a可以延長p53蛋白的半衰期。 / 基於以上實驗事實，我們推測miR-133a誘導p21是通過穩定p53蛋白來實現的。通過數個miRNA靶基因預測軟體，我們判斷RFFL可能是miR-133a發揮效力的直接靶基因。RFFL是E3連接酶，負責非磷酸化p53和磷酸化p53的降解。在RFFL的3側非翻譯區有一個進化保守的miR-133a識別序列。我們克隆此段序列到螢光素酶基因的3側非翻譯區，並進行雙螢光素酶報告基因檢測。測試發現miR-133a可以直接結合到RFFL的此段序列上，並抑制前段基因的翻譯。體外實驗證實，過表達miR-133a可以減少HCT116和HT29細胞內的RFFL蛋白，但並不影響其mRNA。過表達RFFL可以降低p53的表達，反之降低RFFL的蛋白表達，可以提高p53和p21蛋白。上述實驗均有助於證實miR-133a是通過抑制RFFL來提高p21的表達，進而抑制細胞週期。 / 我們也發現miR-133a具有增敏抗癌藥物的效力。單獨轉染miR-133a並不能顯著引發細胞凋亡，而聯合使用miR-133a及抗癌藥物doxorubicin，或者Oxaliplatin都可以顯著增強細胞的早期凋亡。 / 基於上述實驗結果，我們證實miR-133a是一個抑制腫瘤生長的miRNA，其機制可能是通過抑制RFFL蛋白的表達，並啟動p53/p21信號通路引起的。我們的實驗說明miR-133a可以對抗腫瘤藥物起到增敏的作用。 / We found that miR-133a was significantly down-regulated in colorectal cancer (CRC) tissues using miRNA array. However, the role of miR-133a in CRC is largely unknown. We sought to clarify its biologic function, molecular basis, and target gene in CRC. The down-regulation of miR-133a was verified in 94 primary CRC tumours compared with the matched adjacent normal tissues (p < 0.001) and in 9 human colon cancer cell lines. Ectopic expression of miR-133a in colon cancer cell lines (HCT116 and LoVo) significantly suppressed cell growth as evidenced by cell viability assay (p < 0.01 and p < 0.05), and colony formation assay (p < 0.01). Cell cycle analysis revealed that miR-133a caused an increased proportion of cells arrested at G0/G1 phase in HCT116 and LoVo, concomitant with the up-regulation of key G1 phase regulators CDK inhibitors p21 and p27. Promoter-luciferase activity assays indicated that miR-133a markedly increased p53 binding activity and induced p21 transcription. We further revealed that miR-133a decelerated p53 degradation, suggesting that miR-133a was an important positive modulator of the p53/p21 pathway. In silico search showed that the 3’UTR of ring finger and FYVE-like domain containing E3 ubiquitin protein ligase (RFFL), an E3 ubiquitin protein ligase targeting p53 for degradation, contains an evolutionarily conserved miR-133a binding site. Co-transfection with miR-133a repressed RFFL-3’UTR reporter activity for up to 53% (p < 0.01) and remarkably reduced RFFL protein level, indicating that miR-133a directly bound to RFFL mRNA and inhibited RFFL translation. Moreover, miR-133a sensitized colon cancer cells to chemotherapeutic agents (doxorubicin and oxaliplatin) by enhancing apoptosis (p < 0.01) and inhibiting cell proliferation (p < 0.001), adding further weight to the potential significance of miR-133a as a tumour suppressor in inhibiting CRC. In conclusion, miR-133a serves as a functional tumour suppressor in CRC through direct inhibition of the oncogenic RFFL and induction of the p53/p21 pathway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Dong, Yujuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.VI / LIST OF FIGURES --- p.VII / LIST OF TABLES --- p.IX / ABBREVIATIONS --- p.X / TABLES OF CONTENT --- p.XIII / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Colorectal cancer --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.4 / Chapter 1.1.3 --- CRC prevention, screening and therapy --- p.7 / Chapter 1.2 --- microRNA (miRNA) --- p.13 / Chapter 1.2.1 --- Biogenesis of miRNA --- p.13 / Chapter 1.2.2 --- Diagnostic value of miRNAs in CRC --- p.16 / Chapter 1.2.3 --- Prognostic value of miRNAs in CRC --- p.25 / Chapter 1.2.4 --- Predictive value of miRNAs for treatment response in CRC --- p.28 / Chapter 1.2.5 --- miRNA-related single-nucleotide polymorphisms and CRC --- p.29 / Chapter 1.2.6 --- miRNAs and their function in CRC genesis --- p.33 / Chapter 1.2.7 --- Future perspective of miRNAs in CRC --- p.41 / Chapter 1.3 --- Hypothesis and objectives --- p.41 / Chapter CHAPER TWO --- METHODOLGY --- p.43 / Chapter 2.1 --- Cell cultures --- p.43 / Chapter 2.2 --- Patients and clinical specimens --- p.43 / Chapter 2.3 --- miRNA extraction from tissue and cell --- p.46 / Chapter 2.4 --- Micro-dissection and RNA extraction from paraffin sections --- p.47 / Chapter 2.5 --- Real-time quantitative PCR for miRNA microarray --- p.48 / Chapter 2.6 --- miRNA expression analysis --- p.48 / Chapter 2.7 --- mRNA expression analysis --- p.49 / Chapter 2.8 --- RNA interference --- p.52 / Chapter 2.9 --- Colony formation assay --- p.52 / Chapter 2.10 --- MTT cell viability assay --- p.53 / Chapter 2.11 --- Flow cytometry for cell cycle analysis --- p.53 / Chapter 2.12 --- Flow cytometry for cell apoptosis analysis --- p.54 / Chapter 2.13 --- Protein degradation assay --- p.54 / Chapter 2.14 --- Western blot analysis --- p.55 / Chapter 2.15 --- Immunofluorescence staining --- p.56 / Chapter 2.16 --- Plasmids construction --- p.58 / Chapter 2.16.1 --- pRFFL plasmid --- p.58 / Chapter 2.16.2 --- pMIR-RFFL-3’UTR and pMIR-RFFLmut-3’UTR --- p.58 / Chapter 2.17 --- Construction of stable cell lines --- p.59 / Chapter 2.18 --- Dual-luciferase reporter assay for p53 signaling pathway --- p.61 / Chapter 2.19 --- Dual-luciferase reporter assay for RFFL 3’UTR and miR-133a binding activity --- p.62 / Chapter 2.20 --- 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment --- p.62 / Chapter 2.21 --- Tumour xenografts in nude mice model (miRNA intratumoural injection model) --- p.63 / Chapter 2.22 --- Tumour xenografts in nude mice model (miRNA stable cell line subcutaneous injection) --- p.64 / Chapter 2.23 --- Statistical analysis --- p.64 / Chapter CHAPTER THREE --- RESULTS --- p.66 / Chapter 3.1 --- Identification of differentially expressed miRNAs in CRC --- p.66 / Chapter 3.2 --- miR-133a is down-regulated in primary human CRC and colon cancer cell lines --- p.69 / Chapter 3.3 --- Ectopic expression of miR-133a inhibits tumourigenic properties of CRC cells --- p.75 / Chapter 3.3.1 --- miR-133a suppresses cell viability and colony formation --- p.75 / Chapter 3.3.2 --- miR-133a inhibits tumour growth in nude mice --- p.78 / Chapter 3.3.3 --- miR-133a suppresses cell cycle progression --- p.81 / Chapter 3.4 --- G0/G1 phase arrest by miR-133a is mediated through up-regulation of CDKN1A and CDKN1B --- p.83 / Chapter 3.5 --- miR-133a activates p53/p21 pathway through stabilization of p53 protein --- p.88 / Chapter 3.5.1 --- miR-133a induces p21 expression in a p53 wild-type cells --- p.88 / Chapter 3.5.2 --- miR-133a induces p21 promoter transcription activity and p53 binding activity --- p.90 / Chapter 3.5.3 --- Silence of p53 abolished miR-133a induced p21 --- p.92 / Chapter 3.5.4 --- miR-133a increases p53 activity through increase of p53 protein stability --- p.95 / Chapter 3.5.5 --- miR-133a has no effect on c-Myc level --- p.98 / Chapter 3.6 --- miR-133a increases p53 protein level by directly down-regulating RFFL --- p.100 / Chapter 3.7 --- Knock-down of RFFL inhibits cancer cell growth --- p.105 / Chapter 3.8 --- miR-133a sensitized CRC cells to chemotherapeutic drugs treatment --- p.110 / Chapter 3.9 --- Pharmacological demethylation restores miR-133a expression in CRC cells --- p.117 / Chapter 3.10 --- Association between miR-133a expression in tumour and clinicopathological characteristics of CRC patients --- p.119 / Chapter 3.11 --- Validation of other dysregulated miRNAs in CRC --- p.123 / Chapter CHAPTER FOUR --- DISCUSSION --- p.128 / Chapter 4.1 --- Biological role of miR-133a as a tumour suppressor --- p.128 / Chapter 4.2 --- p53/p21 pathway is a critical mediator of miR-133a in CRC --- p.129 / Chapter 4.3 --- Functional significance of RFFL in miR-133a induced p53/p21 signaling --- p.131 / Chapter 4.4 --- Clinical potential of miR-133a in CRC --- p.133 / Chapter 4.5 --- Other dysregulated miRNA --- p.136 / Chapter 4.6 --- Limitations and improvements of the study --- p.136 / Chapter 4.7 --- Conclusions --- p.137 / REFERENCE --- p.139 / PUBLICATIONS --- p.153