宿主防御肽，如抗菌肽和防御素，是固有性免疫的重要组成部分。LL-37是由37个残基组成的阳离子宿主防御肽，是目前唯一被发现的人源宿主防御肽。它在不同的生物过程中都起着关键作用。新证据表明，LL-37与肿瘤进展也有关系。在许多类型的人类恶性肿瘤中它有不同表达，但在结肠癌中的表达和作用，仍未知。在此，我们将对LL-37及其17至32残基片断（ 简称FK-16）对结肠癌的影响进行研究。 / 免疫组化染色结果表明，LL-37在人类结肠癌组织中的表达比正常组织有显著减少。并且，LL-37的表达与TUNEL阳性细胞数量呈正比。合成的LL-37能够诱导不同的结肠癌细胞发生不依赖半胱天冬酶激活的凋亡细胞死亡。并且，LL-37通过激活p53下调Bcl2及上调Bax与Bak来诱导凋亡。LL-37也促使肿瘤坏死因子和核酸内切酶G 向核内转移，以其为目标的siRNA沉默能使细胞对LL-37诱导的凋亡呈现出耐受现象。更重要的是，LL-37的促凋亡作用被发现可以通过对百日咳敏感的Gαi蛋白偶联受体来介导。同时，宿主防御肽敲除的小鼠肠黏膜中，p53、Bax和Bak表达减少而Bcl2表达增加，凋亡的基础水平量也减少。由此说明，LL-37可通过激活GPCR-p53-Bax / Bak / Bcl-2的新信号级联反应来激活AIF / EndoG调控的结肠癌细胞凋亡。 / 与LL-37类似，其片断FK-16也促使不同结肠癌细胞株死亡。但其死亡诱导机制与LL-37不尽相同。FK-16引发了一种独特的死亡方式，即初始诱导不依赖半胱天冬酶激活的凋亡之后紧随引发自噬性细胞死亡。而LL-37没有明显引起这种自噬性死亡。孵育FK-16 24小时后，结肠癌细胞被证明发生凋亡。延长孵育至48小时，细胞的生化和形态学体征符合自噬，包括增加LC3阳性自噬体，积累酸性自噬泡与自溶酶体，和提高 LC3-II水平。敲除两个自噬有关基因 ATG5 和ATG7, 能够部分逆转由FK-16所引起的细胞死亡。并且，细胞凋亡和细胞自噬机制相关信号通路之间存在的交叉调控，在此研究中也被深入提及。 / Host defense peptides, such as cathelicidins (LL-37) and defensins, are important components in the innate immunity. LL-37, a human cationic host defense peptide composed of 37 residues, is the only cathelicidin described so far in humans. It plays a key role in diverse biological processes, including natural immunity, inflammation and tissue repair. Emerging evidence suggests that LL-37 is implicated in cancer development. In this regard, the expression of LL-37 is found to be dysregulated in many types of human malignancy, including lung, breast, ovarian, and gastric cancers. The expression and function of LL-37 in colon cancer, however, are still unknown. In this thesis, the roles of LL-37 and its 17-32 fragment (hereafter referred to as FK-16) in colon cancer development were investigated. / By immunohistochemical staining, it is demonstrated that the expression of LL-37 was significantly reduced in human colon cancer tissues as compared with the cancer adjacent normal tissues. Moreover, LL-37 expression was positively correlated with the number of TUNEL-positive cells. Furthermore, synthetic LL-37 induced caspase-independent apoptotic cell death in different cultured colon cancer cells. In this connection, LL-37 induced apoptosis via downregulation of Bcl-2 and upregulation of Bak and Bax in a p53-dependent manner. It also induced the upregulation and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG), whose targetings by siRNAs rendered the cells resistant to LL-37-induced apoptosis. Above all, the pro-apoptotic effect of LL-37 was found to be mediated through a pertussis toxin-sensitive Gαi protein-coupled receptor. Concordantly, colonic mucosa of cathelicidin-knockout mice exhibited reduced expression of p53, Bax and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Taken together, we demonstrated that LL-37 activates a novel signaling cascade involving the GPCR-p53-Bax/Bak/Bcl-2 axis to activate AIF/EndoG-mediated apoptosis in colon cancer cells. / Similar to the effect of LL-37 peptide, the fragment FK-16 also induced cell death in colon cancer cell lines. However, the action is different. Results demonstrated that FK-16 triggered a unique pattern of cell death characterized by initial caspase-independent apoptosis followed by autophagic cell death, the latter of which was not observed obviously in cells treated with LL-37. Treating colon cancer cells with FK-16 for 24 h induced apoptosis as evidenced by phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Prolonged treatment with FK-16 induced biochemical and morphological features consistent with autophagy, including increased formation of LC3+ autophagosomes, the accumulation of acidic vesicular organelles and autolysosomes, and increased levels of LC3-I/II, Atg5 and Atg7. Knockdown of Atg5 or Atg7 partially reversed the cytotoxic effect of FK-16, suggesting that FK-16-induced autophagy was pro-death in nature. Furthermore, the novel cross-talks between apoptotic and autophagic signalings were also noted. / Collectively, the present study not only contributes to understanding the role of host defense peptide cathelicidin in tumorigenesis, but also provides pre-clinical evidence to propel the development and application of these peptides as novel therapeutic agents for the treatment of colon cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / 综上所述，目前的研究不仅有助于理解宿主防御肽在肿瘤发生, 同时也提供了临床前研究证据，推动了宿主防御肽的开发和应用, 这些肽片段为治疗结肠癌提供了新的治疗手段 。 / Ren, Shunxiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 176-208). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Declaration --- p.vi / Acknowledgements --- p.vii / Publications --- p.ix / Table of contents --- p.xiii / List of illustrations --- p.xviii / Abbreviations --- p.xxiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Colorectal cancer --- p.1 / Chapter 1.1.1 --- Epidemiology of colorectal cancer --- p.1 / Chapter 1.1.2 --- Etiology of colorectal cancer --- p.3 / Chapter 1.1.3 --- Pathogenesis of CRC --- p.8 / Chapter 1.1.4 --- Chemotherapy of colorectal cancer --- p.9 / Chapter 1.2 --- Programmed cell death (PCD) --- p.10 / Chapter 1.2.1 --- Cell death --- p.10 / Chapter 1.2.2 --- Apoptosis --- p.11 / Chapter 1.2.2.1 --- Mechanisms of apoptosis --- p.12 / Chapter 1.2.2.1.1 --- Caspase-dependent apoptosis --- p.13 / Chapter 1.2.2.1.2 --- Caspase-independent apoptosis --- p.15 / Chapter 1.2.2.1.3 --- Tumor suppressor p53 --- p.17 / Chapter 1.2.2.2 --- G-coupled protein receptors in apoptosis --- p.18 / Chapter 1.2.3.1 --- Types of Autophagy --- p.20 / Chapter 1.2.3.2 --- Biological process --- p.22 / Chapter 1.2.3.3 --- Biological functions --- p.24 / Chapter 1.2.3.4 --- Autophagic machinery --- p.27 / Chapter 1.2.3.5 --- Autophagy in cancer --- p.30 / Chapter 1.2.3.6 --- Autophagy and apoptosis --- p.32 / Chapter 1.3 --- Biological functions of cathelicidin --- p.33 / Chapter 1.3.1 --- Antimicrobial activity --- p.34 / Chapter 1.3.2 --- Immunological functions --- p.35 / Chapter 1.3.3 --- Wound healing, angiogenesis and mitogenesis --- p.36 / Chapter 1.3.4 --- Programmed cell death --- p.38 / Chapter 1.4 --- Aim of the present study --- p.39 / Chapter Chapter 2 --- Methods / Chapter 2.1 --- General --- p.41 / Chapter 2.1.1 --- Chemicals and reagents --- p.41 / Chapter 2.1.2 --- Antibodies --- p.44 / Chapter 2.1.3 --- Commercial kits --- p.45 / Chapter 2.1.4 --- Peptide synthesis --- p.46 / Chapter 2.1.5 --- Experimental Animals --- p.46 / Chapter 2.1.6 --- Cell Culture --- p.47 / Chapter 2.2 --- DNA Methylation Analysis --- p.48 / Chapter 2.2.1 --- 5-aza-2’-deoxycytidine (5’Aza-dC) Treatment --- p.48 / Chapter 2.2.2 --- Bisulfite Genomic Sequencing and Methylated-DNA capture (MethylCap)-qPCR --- p.48 / Chapter 2.3 --- Effects of LL-37 and its analogue FK-16 in colon cancer cells in vitro --- p.48 / Chapter 2.3.1 --- Cell viability Assay --- p.49 / Chapter 2.3.2 --- Lactic dehydrogenase (LDH) activity --- p.49 / Chapter 2.3.3 --- Cell cycle analysis --- p.49 / Chapter 2.3.4 --- Measurement of apoptosis in vitro --- p.50 / Chapter 2.3.4.1 --- Quantitation of DNA fragmentation --- p.50 / Chapter 2.3.4.2 --- Quantitation of phosphatidylserine externalization --- p.50 / Chapter 2.3.5 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.3.6 --- Nuclear protein extraction --- p.52 / Chapter 2.3.7 --- Western Blot --- p.53 / Chapter 2.3.8 --- Immunofluorescence --- p.54 / Chapter 2.3.9 --- Bcl-2 overexpression --- p.54 / Chapter 2.3.9.1 --- Transforming competent cells --- p.55 / Chapter 2.3.9.2 --- Plasmid DNA purification --- p.55 / Chapter 2.3.10 --- RNA interference --- p.56 / Chapter 2.3.11 --- Detection of acidic vesicular organelles (AVOs) with acridine orange --- p.57 / Chapter 2.3.12 --- Labeling of autophagic vacuoles with monodansylcadaverine (MDC) --- p.58 / Chapter 2.3.13 --- Transmission electron microscopy --- p.59 / Chapter 2.4 --- Cathelicidin-knockout (Cnlp/) mice model --- p.60 / Chapter 2.4.1 --- Normal mouse colon sample collection --- p.60 / Chapter 2.4.2 --- Tissue Processing --- p.61 / Chapter 2.4.3 --- Measurement of basal apoptosis in normal colon tissues --- p.61 / Chapter 2.5 --- Clinical samples --- p.62 / Chapter 2.5.1 --- Immunohistochemistry of clinical samples --- p.62 / Chapter 2.5.2 --- Measurement of colonocyte apoptosis of clinical samples --- p.62 / Chapter 2.5.3 --- Evaluation of colonocyte proliferation of clinical samples --- p.63 / Chapter 2.6 --- Statistical analysis --- p.63 / Chapter Chapter 3 --- Results and Discussion / Chapter 3.1 --- LL-37 was downregulated in colon cancer tissues --- p.64 / Chapter 3.2 --- Effects of LL-37 on human colon cancer cells --- p.72 / Chapter 3.2.1 --- LL-37 induced DNA fragmentation and phosphatidylserine externalization without caspase activation in colon cancer cells --- p.72 / Chapter 3.2.2 --- LL-37 induced AIF- and EndoG-dependent apoptosis --- p.79 / Chapter 3.2.3 --- Altered expression of Bcl-2 family members was required for AIF- and EndoG-mediated apoptosis induced by LL-37 --- p.84 / Chapter 3.2.4 --- p53 activation was required for LL-37-induced apoptosis --- p.90 / Chapter 3.2.5 --- The apoptogenic action of LL-37 was mediated by G protein-coupled receptor (GPCR) --- p.94 / Chapter 3.2.6 --- Reduced basal apoptotic rate in colonic mucosa of cathelicidin-knockout mice --- p.95 / Chapter 3.2.7 --- Preliminary Discussion --- p.103 / Chapter 3.3 --- Effects of FK16 on human cancer cells --- p.108 / Chapter 3.3.1 --- FK-16 induced AIF- and EndoG-dependent apoptosis in colon cancer cells --- p.108 / Chapter 3.3.2 --- FK-16 induced autophagic cell death in colon cancer cells --- p.116 / Chapter 3.3.3 --- Activation of p53 was required for FK-16-indcued apoptosis and autophagy cell death --- p.123 / Chapter 3.3.4 --- Altered expression of Bcl-2 and Bax was required for FK-16-indcued apoptosis and autophagic cell death --- p.129 / Chapter 3.3.5 --- FK-16-induced apoptosis and autophagic cell death were reciprocally regulated --- p.134 / Chapter 3.3.6 --- Preliminary Discussion --- p.139 / Chapter Chapter 4 --- Summary and Finial Conslusion --- p.142 / References --- p.144

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

目的：結直腸癌是世界上第三常見惡性腫瘤，結腸鏡檢查是診斷的金標准。但其創傷性、昂貴的設備以及人力的需求阻礙了廣泛應用。本研究評估了糞便miRNA作為非損傷性分子生物標記物篩查結直腸腺瘤和腫瘤的可行性，並深入探究了致癌miRNA的基因靶點。 / 方法：我們評估了糞便miRNAs的穩定性以及檢測的可重復性。糞便樣本收集自88例結腸直腸癌患者，57例結直腸息肉患者和101名健康對照，用實時定量逆轉錄PCR檢測miRNA水平。所有候選miRNA標記物在配對的癌及癌旁組織中進行驗証。我們共測試了糞便中7種miRNAs，包括前期報道在結直腸癌中上調的miR-21和miR-92a(第一部分)，以及在667個miRNA中在結直腸癌上調最高的5個miRNA(第二部分)。我們研究了它們的水平與腫瘤分期及位置的關系。並隨訪了病人經腫瘤或腺瘤切除術后其糞便miRNA水平，從而証實它們是否與腫瘤相關。我們應用了彗星試驗、細胞活力試驗、集落形成試驗，以及細胞凋亡分析試驗研究了miR-18a在腫瘤的發展過程中的作用(第三部分)。 / 結果：第一部分，我們確定糞便miRNA的穩定性，能被實時定量逆轉錄PCR檢測並顯示高重復性。糞便miR-92a標記物的靈敏度和特異性分別為71.6%和73.3%，miR-21分別為55.7%和73.3%。MiR-92a水平顯示遠端結直腸癌比近端結直腸癌的檢測具有更高靈敏度，晚期腺瘤比小息肉更具靈敏度。腫瘤切除后，miR-21和miR-92a水平顯著下降。 / 第二部分，基於結直腸腫瘤miRNA的表型，我們發現糞便miR-18a, miR-20a, miR-135b和miR-221能作為標記物鑒別結直腸癌，miR-18a (敏感度: 51.1%, 特異性: 90.1%); miR-20a (72.7%, 81.2%) ; miR-135b (81.8%, 68.3%); miR-221 (69.3%, 77.2%)。腫瘤切除后，這四種標記物會顯著下降。MiR-135b和miR-221也能鑒別腺瘤。四種標記物對遠近端結腸癌的檢測無顯著差異。 / 第三部分，通過程序和熒光素酶報告基因活性預測和驗証，我們發現一種重要的DNA修復蛋白---共濟失調毛細血管擴張突變(ATM)是miR-18a的靶蛋白。MiR-18a的異位表達減弱細胞DNA雙鏈損傷修復機制，導致腫瘤發生的易感性。 / 結論：我們發現糞便中miR-21, miR-92a, miR-18a, miR-20a, miR-135b 和miR-221標記物能夠鑒別結直腸癌。MiR-92a, miR-135b 和miR-221能鑒別結直腸腺瘤。MiR-18a抑制共濟失調毛細血管擴張突變基因表達並減弱細胞DNA雙鏈損傷修復機制。糞便miRNA是結直腸癌篩查的有效生物標記物。 / Objective: Colorectal cancer (CRC) is the third most common cancer worldwide. Colonoscopy is the current gold standard for diagnosing CRC. However, its invasive nature, the cost of equipment and the demand for manpower have hampered the wide application of this procedure. This study evaluated the feasibility of using stool-based miRNA as non-invasive biomarkers for the screening of colorectal adenoma and cancer, and investigated the gene target of a candidate oncogenic miRNA. / Methods: The reproducibility of detection and stability of stool-based miRNAs were first evaluated. Stool samples were collected from 88 CRC patients, 57 patients with colorectal polyp and 101 healthy controls MiRNA levels were detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). AII candidate miRNA markers were validated in a cohort of paired tumor and adjacent normal tissues. In total, we tested 7 miRNAs in the stool, including miR-21 and miR-92a which were reported to be up-regulated in CRC in previous studies (part one), and 5 miRNAs which were found to be the most up-regulated in colorectal tumor based on the profiling of 667 miRNAs (part two). Their levels with tumor stage and location were evaluated. Their change in level was followed up in a subset of patients after the removal of tumor or adenoma. We investigated miR-18a for its function in cancer development using comet assay, cell viability assay, colony formation assay, and analysis on apoptosis (part three). / Results: In part one, we found stool miRNAs stable and detectable with high reproducibility by qRT-PCR. In detecting CRC, stool miR-92a had a sensitivity of 71.6% and a specificity of 73.3%, stool miR-21 had a sensitivity of 55.7% and a specificity of 73.3%. Stool miR-92a level had higher sensitivity for distal CRC than proximal CRC, and a higher sensitivity for advanced adenoma than minor polyps. The removal of tumor resulted in reduced stool miR-21 and miR-92a levels. / In part two, based on miRNA profiling of CRC tumors, we found that stool-based miR-18a, miR-20a, miR-135b, and miR-221 can discriminate colorectal cancer patients from healthy individuals: miR-18a (sensitivity: 51.1%, specificitiy: 90.1%); miR-20a (72.7%, 81.2%); miR-135b (8 1.8%, 68.3%); miR-221 (69.3%, 77.2%). Levels of these 4 stool-based markers dropped after removal of tumors. Stool-based miR-135b and miR-221 also discriminated patients with adenoma from healthy individuals. MiR-18a, miR-20a, miR-135b and miR-221 showed no desparity in detecting proximal or distal colon cancer. / In part three, based on in silico prediction and validation with luciferase reporter activity, we identified Ataxia Telangiectasia Mutated (ATM ), a protein crucial to DNA repair, as a target of miR-18a. Ectopic expression miR-18a attenuates DNA double strand break repair mechanism, creating a genetic predisposition to the development of cancer. / Conclusion: Stool-based miR-21, miR-92a, miR-18a, miR-20a, miR-135b and miR-221 can discriminate patients with CRC from healthy individuals. Notably, a subset of these miRNAs (miR-92a, miR-135b, and miR-221) can discriminate patients with colorectal adenoma from healthy individuals MiR-18a suppressed ATM gene expression and attenuated cellular repair mechanism to DNA double strand breaks. Stool-based miRNAs are useful CRC screening biomarkers. / 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. / Wu, Chung Wah. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 80-92). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Colorectal cancer --- p.1 / Chapter 1.2 --- Current screening methods --- p.3 / Chapter 1.2.1 --- Colonoscopy --- p.3 / Chapter 1.2.2 --- Sigmoidoscopy --- p.4 / Chapter 1.2.3 --- Stool-based tests --- p.4 / Chapter 1.2.3.1 --- Fecal occult blood test --- p.5 / Chapter 1.2.3.2 --- Stool-based DNA test --- p.6 / Chapter 1.2.3.3 --- Stool-based RNA and protein test --- p.7 / Chapter 1.3 --- MiRNA and its role in cancer --- p.8 / Chapter 1.4 --- Aims of study --- p.9 / Chapter CHAPTER TWO --- METHODOLOGY --- p.10 / Chapter 2.1 --- Subjects and sample collection --- p.10 / Chapter 2.2 --- MiRNA extraction in tissue and stool samples --- p.13 / Chapter 2.3 --- MiRNA quantitation by quantitative reverse transcription quantitative reverse transcription polymerase chain reaction --- p.14 / Chapter 2.4 --- Determining the stability of miRNA in stool samples --- p.15 / Chapter 2.5 --- Determining the reproducibility of miRNA quantitation in stool samples --- p.16 / Chapter 2.6 --- Reverse transcription for miRNA array --- p.16 / Chapter 2.7 --- Quantitative polymerase chain reaction for miRNA array --- p.16 / Chapter 2.8 --- Cell culture, miRNA precursors and transfection --- p.17 / Chapter 2.9 --- Dual-luciferase reporter assay --- p.17 / Chapter 2.10 --- Quantitative reverse transcription polymerase chain reaction for mRNA --- p.19 / Chapter 2.11 --- Western blot analysis --- p.19 / Chapter 2.12 --- Comet assay --- p.19 / Chapter 2.13 --- Colony formation and cell viability assay --- p.20 / Chapter 2.14 --- Annexin V apoptosis assay --- p.21 / Chapter 2.15 --- Statistics --- p.21 / Chapter CHAPTER THREE --- RESULTS --- p.23 / Chapter 3.1 --- PART 1 --- p.23 / Chapter 3.1.1 --- Stability of miRNA detection in stool samples --- p.23 / Chapter 3.1.2 --- Reproducibility of miRNA quantitation in stool samples --- p.23 / Chapter 3.1.3 --- Detection and normalization of miRNA levels --- p.25 / Chapter 3.1.4 --- Expression of miR-21 and miR-92a in CRC tissue samples --- p.28 / Chapter 3.1.5 --- Levels of stool-based miR-21 and miR-92a in CRC and polyp patients --- p.30 / Chapter 3.1.6 --- Sensitivity of stool-based miR-21 and miR-92a towards colorectal cancer and polyps --- p.32 / Chapter 3.1.7 --- Association of stool-based miR-21 and miR-92a with clinicopathological features --- p.34 / Chapter 3.1.8 --- Follow-up on stool miR-21 and miR-92a levels after removal of lesion --- p.37 / Chapter 3.2 --- Part 2 --- p.39 / Chapter 3.2.1 --- MiRNA profiling in colorectal tumors --- p.39 / Chapter 3.2.2 --- Validation of miRNA profiling results --- p.41 / Chapter 3.2.3 --- Candidate miRNA levels in stool samples of CRC and adenoma patients --- p.44 / Chapter 3.2.4 --- Sensitivities and specificities of miRNA candidates for adenoma and CRC --- p.47 / Chapter 3.2.5 --- Sensitivties of miRNA candidates based on tumor location --- p.49 / Chapter 3.2.6 --- Follow-up on stool miRNA levels after removal of lesion --- p.51 / Chapter 3.2.7 --- Association of stool-based miRNAs with nodal involvement in CRC --- p.53 / Chapter 3.2.8 --- Establishing the miRNA marker panel --- p.55 / Chapter 3.3 --- Part 3 --- p.57 / Chapter 3.3.1 --- In Silico prediction of miR-18a target and validation by luciferase assay --- p.57 / Chapter 3.3.2 --- Expression and correlation between miR-18a and ATM in paired colorectal tumor tissue, cell lines and normal colon biopsies --- p.60 / Chapter 3.3.3 --- Regulation of double strand DNA damaga recovery by miR-18a --- p.62 / Chapter 3.3.4 --- Cell sensitization to genotoxin by miR-18a --- p.64 / Chapter 3.3.5 --- Effect of miR-18a on genotoxin induced apoptosis --- p.66 / Chapter CHAPTER FOUR --- DISCUSSION --- p.68 / Chapter 4.1 --- Stability and detection reproducibility of stool-based miRNA --- p.68 / Chapter 4.2 --- Stool-based miRNAs for screening colorectal cancer and polyps/adenomas --- p.69 / Chapter 4.2.1 --- MiR-21 --- p.69 / Chapter 4.2.2 --- MiR-18a, miR-20a and miR-92a --- p.70 / Chapter 4.2.3 --- MiR -135b and miR -221 --- p.71 / Chapter 4.2.4 --- MiR -31 --- p.72 / Chapter 4.3 --- Discriminating proximal and distal CRC --- p.73 / Chapter 4.4 --- Evaluation of stool-based miRNA level after removal of lesions --- p.74 / Chapter 4.5 --- MiRNA marker panel --- p.74 / Chapter 4.6 --- Advantages of stool-based miRNA tests --- p.75 / Chapter 4.7 --- Ataxia telangiectasia mutated as the direct target of miR -18a --- p.75 / Chapter 4.8 --- Future directions for study --- p.78 / Chapter 4.9 --- Conclusion --- p.78 / REFERENCES --- p.80 / PUBLICATIONS --- p.93

Colorectal cancer is one of the commonest malignancies worldwide. Its prevention, diagnosis and treatments have attracted multidisciplinary attention. Surgery is the mainstay of treatment for colorectal cancer. It was estimated that up to 85% of colorectal cancer were amenable to surgical treatment, whether curative or palliative. Not surprisingly laparoscopic resection of colorectal cancer was reported soon after cholecystectomy. However, with the appearance of early port site recurrence, most authorities were concerned about the adequacy of tumour clearance and the long-term survival after laparoscopic resection. / In this thesis, comparative and randomized studies were conducted to answer the above questions. It was concluded that, as compared to conventional open surgery, laparoscopic assisted resection of recto-sigmoid carcinoma was less painful and allowed earlier post-operative recovery. Tissue trauma, as reflected by systemic cytokines response, was less after laparoscopic assisted resection. Some cellular components of immune system were also less suppressed. Most importantly, laparoscopic resection did not jeopardize the survival and disease control of patients. The justification of adopting laparoscopic technique would depend on the societal value of its effectiveness in improving the short-term post-operative outcomes. / Laparoscopic technology and its application may be the biggest advancement in nearly all surgical specialties in the last decade. Since the introduction of laparoscopic cholecystectomy, enthusiastic surgeons have attempted laparoscopic approach in almost every type of operations, and many of the techniques have gained public acceptance within a very short time. However, most of these developments were not based on good scientific evidence from comparative study. While laparoscopic cholecystectomy was shown to cause less pain and allow patients to recover earlier after operation, these benefits may or may not be conferred to other procedures and diseases. / Therefore, to justify the use of laparoscopic assisted colorectal resection for carcinoma, two criteria must be satisfied. Firstly the long term survival and the disease free interval of patients should not be adversely affected, as these are the most important endpoints in the success of tumour surgery. Secondly, the proposed benefits of minimally invasive surgery must be demonstrated, otherwise it is not worthwhile to adopt a new technique. / Leung Ka Lau. / "July 2005." / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0174. / Thesis (M.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 122-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

Introduction: Gastric and colon cancer are major causes of mortality and morbidity worldwide. Gastric cancer is often detected at an advanced stage and current chemotherapeutics are only modestly effective against this neoplasm. Novel chemotherapeutics, chemopreventive agents and treatment strategies are required to prevent and treat gastric cancer. The ideal method to eliminate cancer cells may be the induction of apoptosis, further preventing cell proliferation and tumour growth. Recently, short-chain fatty acids (SCFAs) butyrate and propionate have been investigated as potential chemotherapeutic agents, particularly in colon cancer. Butyrate is reported to induce apoptosis in colon cancer cells and is demonstrated to modulate intracellular redox state by altering the levels of an antioxidant, glutathione (GSH). GSH availability is controlled by the oxidative pentose pathway (OPP). Very few studies have investigated the effects of butyrate on cell types other than colon cancer cells, and even less is known regarding the effects of propionate. This thesis investigated the potential for SCFAs to induce apoptosis in a gastric cancer cell line, Kato III, compared to the colon cancer cell line, Caco-2. Cell cycle regulation, OPP activity, GSH availability and glucose metabolism were also assessed. Methods: Initial studies developed a new technique to measure 1-13C-D-glucose metabolism. Following this, Kato III and Caco-2 colon carcinoma cells were treated with butyrate or propionate (1mM, 5mM or 10mM) or a 5mM combination of both SCFAs. The induction of apoptosis and cell cycle alterations by these SCFAs were assessed using flow cytometry. OPP activity and GSH availability were assessed in both cell lines using colorimetric techniques. Butyrate metabolism was assessed using 13C-butyrate. Results: Butyrate and propionate significantly induced apoptosis and G2-M arrest in Kato III and Caco-2 cells, although to a significantly greater extent in the latter cell line. Moreover, butyrate induced apoptosis to a significantly greater extent than propionate, in both cell lines. SCFA treatment led to the significant up-regulation of OPP activity in both cancer cell lines while GSH availability was significantly reduced. Glucose metabolism was initially increased by all SCFA treatments, however, 72hr butyrate treatment led to its reduction. Importantly, glucose metabolism was measured using a new technique developed within this thesis. The rate of butyrate metabolism was demonstrated to correlate with the sensitivity of each cell line to this SCFA. Conclusions: This thesis provides evidence that SCFAs, particularly butyrate, induce apoptosis in gastric and colon cancer cells in vitro. The response of cancer cells to SCFAs appears complex, and involves multiple distinct mechanisms and pathways, including p53, Fas, changes to intracellular redox state and glucose metabolism. The capability of butyrate to induce apoptosis also appears to be directly related to the rate of its metabolism. Butyrate has the potential to be utilised as an adjunctive therapy for the treatment of gastric cancer and colon cancer. / Thesis (Ph.D.) -- School of Molecular and Biomedical Science, 2007

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

The number of patients with colorectal cancer had increased dramatically in recent years (Hong Kong cancer registry, 2012), and surgical resection is the first line treatment of colorectal. To enhance patients’recovery process, there is a need to develop a comprehensive and user-friendly, with most important, an evidence-based guideline for promoting patients’ recovery process. Traditional post-operative management is associated with different postoperative complications, delayed recovery, and lengthened hospital stay. Recent research documented that using a specific rehabilitation programme focused on education; early mobilization and early diet regime could enhance patients’ recovery. Therefore, this transitional research aims to evaluate the current evidence on the effect of adopting a specific rehabilitation programme, to formulate an evidence-based guideline, assess its implementation potential, and to develop an implementation and evaluation plan. Ten related literature were retrieved from four electronic bibliographical databases. Critical appraisal had been done to ensure the quality and validity of the selected evidences. A clinical guideline is developed based upon the information from the identified high level of literature. The implementation potential is assessed based on the similarity and the readiness of the target setting to the proposed environment. It was found that the transferability of the protocol was high and it was feasible to be implemented into the target site. Little expenditure and input was expected, as the protocol was a systematic reformation of practice, rather that developing a set of totally new practice to current clinical setting. An implementation plan was then planned, which included the communication plan with all the stakeholders. After reaching a consensus among the stakeholders, a two-month pilot study will be carried out for examining the readiness before the full-scale implementation of the program. The evaluation plan of the effectiveness of the proposed program is developed. Result will be used to provide recommendation for further adjustment on the protocol to yield a better outcome. The implementation of this nurse-led rehabilitation program is suggested to be worthy of adoption in the clinical setting for bringing benefits to patients, the hospital and staffs. / published_or_final_version / Nursing Studies / Master / Master of Nursing

MicroRNAs (miRNAs) are a small non-coding RNAs that play a crucial role in cellular proliferation, differentiation and apoptosis by regulating gene expression via post-translation repression or degradation. They are involved in the regulation of various human diseases including colorectal cancer (CRC). CRC is the third most common cancer in the world and second leading cause of cancer death in Hong Kong and United States. Distant metastasis is the main cause of high mortality rate. In most CRC patients, liver is the most common site of distant metastasis, which is often associated with treatment failure and poor prognosis. While some patients with liver metastasis may still be amenable to surgical resection, most patients can only be treated with chemotherapy, which has limited in controlling the tumor progression. The pathological mechanism of metastasis in CRC is poorly understood. Cell motility is important for tumor invasion and metastasis, which require the interaction between tumor cells and their extracellular matrix. This interaction is regulated by the focal adhesion molecules. Recent evidence suggested that miRNAs affect the cell motility and invasiveness of various cancers, and regulate key steps in metastatic cascade. Investigation of the role of miRNAs in tumor development and metastasis can provide potential novel targets for treatment of colorectal liver metastasis or even other advanced cancers. In this study, expression of miR-885-5p was examined in CRC surgical specimens. Overexpression of miR-885-5p was observed in liver metastasis when compared with primary tumor and adjacent non-tumorous colon. The high expression level of miR-885-5p in primary colorectal tumors was positively associated with late TNM stage and development of metastasis, suggesting that its expression level can act a predictive marker for liver metastasis. Functional studies demonstrated that overexpression of miR-885-5p could significantly enhance the invasive phenotypes and metastatic properties of colorectal cancer cells through a series of in vitro and in vivo assays. Overexpression of miR-885-5p was correlated with increased expression of mesenchymal markers such as N-cadherin, vimentin and Snail, and decreased expression of epithelial marker such as E-cadherin. Moreover, overexpression of miR-885-5p enhanced the expression of Rho GTPases, which is a regulator of polarity, protrusion and adhesion. These results suggested that miR-885-5p overexpression might be a key step in tumor progression and metastasis via regulation of EMT pathway and Rho GTPases family. Knockdown of miR-885-5p enhanced chemosensitivity of CRC cells through induction of apoptosis. On the other hand, overexpression of miR-885-5p increased tumor proliferation through upregulation of the expression level of cyclin D1. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy