He, Jun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 136-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

大腸癌是全世界最常見的癌症之一，亦是一個癌症死亡率的首要原因。大腸癌患者約50%在病程中會出現轉移病灶。近十年來，雖然多種被批准用於臨床治療的新化療藥顯著提高了大腸癌的治療效果，但是轉移性大腸癌病人的預後仍然很差。隨著各種分子生物技術的進步，新的治療標靶可能在大腸癌細胞株中被發現，並得以在病人標本中驗證。 / 在本研究中，我們採用即時定量多聚酶鏈反應(qPCR)陣列分析，比較大腸癌細胞株和正常大腸細胞株基因表達譜，試圖識別潛在的新的治療標靶。結果提示，與正常大腸細胞株 CCD-18Co 比較，Jagged 2 (JAG2) 和 Frizzled-3 (FZD3)基因 在大腸癌細胞株 SW480 和 SW620 中表達升高。病人大腸癌組織的免疫組織化學染色 (IS) 檢查進一步證實了上述結果，大腸癌組織較其癌旁正常組織表達3.1倍JAG2和6.6倍FZD3蛋白。因此， 我們假設JAG2和FZD3在大腸癌的發生中起重要作用。 / 為了檢驗該假設的真偽，我們運用RNA 干擾的方法進行功能缺失研究。通過該方法，大腸癌細胞株中JAG2 信使RNA和蛋白均能夠被下調，但是FZD3蛋白卻沒有顯示降低。為了弄清JAG2基因的功能，我們進行了單層細胞劃痕傷口癒合試驗和Matrigel 侵襲試驗。結果提示，JAG2 基因下調顯著抑制大腸癌細胞遷移和侵襲的能力。 / 為了調查參與上述功能的機制，我們利用腫瘤轉移相關基因的qPCR陣列分析，試圖檢測出JAG2基因敲除後上調或下調表達的轉移相關基因。結果顯示組織蛋白酶K (CTSK)，一種溶酶體半胱氨酸蛋白酶，在JAG2基因沉默的大腸癌細胞株中表達下調。為了闡明CTSK 活性在大腸癌細胞株侵襲能力中起到的作用，我們採用CTSK抑制劑處理大腸癌細胞株HCT116和DLD-1，發現這兩種細胞株的侵襲能力分別下降了36%和59%。總之, 這些發現表明CTSK可能是JAG2的下游靶基因，活性CTSK可能參與了JAG2介導的大腸癌細胞株侵襲能力。 / 以前的研究表明p38 MAPK通路參與癌細胞遷和侵襲能力的調控。通過Western blot方法，磷酸化的p38和磷酸化的STAT3被發現在JAG2基因沉默的大腸癌細胞中表達降低。p38抑制劑處理的 HCT116和DLD-1細胞降低了侵襲能力下降，同時遷移能力也由於p38抑制劑的處理而降低，支持p38可調控癌細胞遷移和侵襲能力的事實。 / 總之，我們的結果顯示JAG2高表達通過啟動CTSK和p38 MAPK通路，可能促進大腸癌轉移。因此，JAG2可能成為轉移性大腸癌治療的潛在標靶。 / Colorectal cancer (CRC) is one of the most frequent cancers worldwide and is a leading cause of cancer mortality. Around 50% of patients with CRC will experience metastases. Although significant progress has been made in CRC treatment within the last decade with the approval of multiple new chemotherapeutic agents, the prognosis for patients with metastatic CRC remains poor. With the advancement of molecular techniques, novel therapeutic targets are able to be discovered in CRC cell lines and validated in patient samples. / Therefore in this project, I aim to identify potential novel therapeutic targets by comparing the gene expression profile of colon cancer cell lines and a normal colon cell line using quantitative polymerase chain reaction (qPCR) arrays. Results showed that Jagged 2 (JAG2) and Frizzled-3 (FZD3) were up-regulated in the CRC cell lines SW480 and SW620 as compared to the normal colon cell line CCD-18Co. Those results were further validated by immunohistochemical staining (IS), which detected up-regulated JAG2 (3.1-fold) and FZD3 (6.6-fold) proteins expression in CRC tissues as compared to adjacent normal tissues. Thus I hypothesized that JAG2 and FZD3 may play an important role in CRC carcinogenesis. / In order to study the roles of FZD3 and JAG2 in CRC, loss-of-function studies by RNA interference (RNAi) were carried out. While the expression of FZD3 protein failed to be down-regulated by RNAi, JAG2 expression was successfully knocked down in CRC cell lines at both the mRNA and protein levels. Functional analyses using the monolayer scratch wound-healing assay and Matrigel invasion assay showed that JAG2 knockdown significantly inhibited migration and invasion in CRC cell lines. / To investigate the mechanisms involved, a tumour metastasis qPCR array was used to examine the changes in the expression level of metastasis-related genes after JAG2 gene knockdown. Results showed that the expression of Cathepsin K (CTSK), a lysosomal cystein protease, was found to be down-regulated in CRC cell lines following JAG2 silencing. To demonstrate the importance of CTSK activity in CRC cell invasion, HCT116 and DLD-1 CRC cell lines were treated with a CTSK inhibitor and its effect were assessed by the Matrigel invasion assay. Results showed that CTSK inhibition led to a 36% and 59% reduction in number of invaded cells in HCT116 and DLD-1 cell lines, respectively. Taken together, these findings show that CTSK may be a downstream target of JAG2 and that active CTSK may involve in JAG2 mediated invasion in CRC cell lines. / Previous works by others have shown that the p38 MAPK pathway is involved in the regulation of migration and invasive activity of cancer cell lines. Using Western blot analysis, the expression of phosphorylated p38 MAPK and phosphorylated STAT3 were found to be down-regulated following JAG2 depletion in CRC cell lines. In support of a role for p38 MAPK in the regulation of cancer cell migration and invasive capability, treatment with a p38 MAPK inhibitor was found to reduce the percentage of invasive cells and distance moved by migratory cells in HCT116 and DLD-1 cell lines. / In conclusion, my results show that JAG2 over-expression in CRC may promote cancer cell migration and invasion through activation of CTSK and the p38 MAPK pathway. Therefore, JAG2 may be a potential therapeutic target for treatment of metastatic CRC. / 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. / He, Wan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 164-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Colorectal Cancer (CRC) --- p.1 / Chapter 1.1.1 --- Epidemiology and Incidence --- p.1 / Chapter 1.1.2 --- Histology --- p.2 / Chapter 1.1.3 --- Gender and Age --- p.4 / Chapter 1.1.4 --- Etiology of CRC --- p.4 / Chapter 1.1.4.1 --- Environment --- p.4 / Chapter 1.1.4.2 --- Hereditary Factors --- p.5 / Chapter 1.1.4.3 --- Dietary Factors --- p.6 / Chapter 1.1.4.4 --- Obesity --- p.6 / Chapter 1.1.4.5 --- Tobacco and alcoho --- p.7 / Chapter 1.1.4.6 --- Inflammatory bowel disease (IBC) --- p.7 / Chapter 1.1.5 --- Genetic Changes in CRC --- p.8 / Chapter 1.1.5.1 --- Chromosomal Aberration --- p.8 / Chapter 1.1.5.2 --- Tumor Suppressor Genes --- p.10 / Chapter 1.1.5.2.1 --- APC gene --- p.10 / Chapter 1.1.5.2.2 --- P53 gene --- p.11 / Chapter 1.1.5.2.3 --- SMAD4 gene --- p.11 / Chapter 1.1.5.3 --- Oncogenes --- p.12 / Chapter 1.1.5.3.1 --- Epidermal Growth Factor Receptor (EGFR) gene --- p.12 / Chapter 1.1.5.3.2 --- RAS gene and BRAF gene --- p.13 / Chapter 1.1.5.4 --- Proposed Two-hit Model for the Multistep Pathogenesis of CRC --- p.15 / Chapter 1.1.6 --- Clinical Presentation and Diagnosis --- p.16 / Chapter 1.1.7 --- Theatment --- p.16 / Chapter 1.1.7.1 --- Surgery --- p.16 / Chapter 1.1.7.2 --- Radiotherapy (RT) --- p.17 / Chapter 1.1.7.3 --- Concurrent Chemotherapy --- p.17 / Chapter 1.1.7.4 --- Target Therapy --- p.18 / Chapter 1.1.7.5 --- Colorectal Cancer Treatment by Stage --- p.19 / Chapter 1.1.7.6 --- Novel Strategies --- p.20 / Chapter 1.1.7.6.1 --- Epigenetic therapy --- p.20 / Chapter 1.1.7.6.2 --- Immunotherapy --- p.21 / Chapter 1.2 --- Pathways Involved in CRC Carcinogenesisand Progression --- p.22 / Chapter 1.2.1 --- Wnt Signaling Pathway --- p.22 / Chapter 1.2.2 --- Notch Signaling --- p.23 / Chapter 1.2.3 --- Nuclear Factor-kappa B (NF-κB) Signaling Pathway --- p.23 / Chapter 1.2.4 --- Phosphatidylinositol 3-kinase (PI3K) Signaling Pathway --- p.24 / Chapter 1.2.5 --- Crosstalk Among WNT, NOTCH, NF-κB and PI3K Signaling Pathway in CRC --- p.24 / Chapter 1.3 --- Hypothesis and Objectives of this Study --- p.28 / Chapter Chapter 2 --- Identification of Differentially Expressed Genes between Colorectal Cancer Cell Lines and A Normal Colon Cell Line --- p.29 / Chapter 2.1 --- Background --- p.29 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Cell Lines --- p.33 / Chapter 2.2.2 --- Identification of Differetially Expressed Genes by qPCR Arrays --- p.33 / Chapter 2.2.2.1 --- Total RNA Extraction --- p.33 / Chapter 2.2.2.2 --- RNA Quality Contol --- p.34 / Chapter 2.2.2.3 --- Reverse Transcription (RT) --- p.34 / Chapter 2.2.2.4 --- PCR Arrays --- p.34 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Differentially Expressed Genes in WNT Signaling Pathway --- p.36 / Chapter 2.3.2 --- Differentially Expressed Genes in Notch Signaling Pathway --- p.40 / Chapter 2.3.3 --- Differentially Expressed Genes in NF-κB Signaling Pathway --- p.43 / Chapter 2.3.4 --- Differentially Expressed Genes in PI3K-AKT Signaling Pathway --- p.46 / Chapter 2.3.5 --- Choice of over-expressed genes for further validation and characterization --- p.49 / Chapter 2.4 --- Discussions --- p.53 / Chapter 2.4.1 --- WNT Signaling Pathway --- p.53 / Chapter 2.4.2 --- NOTCH Signaling Pathway --- p.54 / Chapter 2.4.3 --- NF-κB Signaling Pathway --- p.55 / Chapter 2.4.4 --- PI3K-AKT Signaling Pathway --- p.56 / Chapter 2.4.5 --- Choice of over-expressed genes for further validation and characterization --- p.56 / Chapter Chapter 3 --- JAG2, FZD3 and NOTCH4 Expression in Colorectal Cancer Cell Lines and Colorectal Cancer Tissues --- p.59 / Chapter 3.1 --- Background --- p.59 / Chapter 3.1.1 --- JAG2 Ligand --- p.59 / Chapter 3.1.2 --- FZD3 Receptor --- p.61 / Chapter 3.1.3 --- NOTCH4 Receptor --- p.62 / Chapter 3.2 --- Materials and Methods --- p.64 / Chapter 3.2.1 --- CRC Cell Lines --- p.65 / Chapter 3.2.2 --- CRC Tissues --- p.65 / Chapter 3.2.3 --- Quantitative RT-PCR --- p.66 / Chapter 3.2.4 --- Detection of JAG2, FZD3 and NOTCH4 Protein Expression in CRC Tissues by Immunohistochemical Staining (IS) --- p.67 / Chapter 3.2.5 --- Western Blot Assays --- p.68 / Chapter 3.2.5.1 --- Protein extraction --- p.68 / Chapter 3.2.5.2 --- SDS-PAGE gel electrophroresis --- p.68 / Chapter 3.2.5.3 --- Protein blotting --- p.68 / Chapter 3.2.6 --- Detection of JAG2 and FZD3 Protein Expression in CRC and Normal Colon Cell Lines by Western Blotting --- p.69 / Chapter 3.2.7 --- Statistical Analysis --- p.70 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- JAG2 and FZD3 but not NOTCH4 mRNA were Over -expressed in CRC Cell Lines --- p.71 / Chapter 3.3.2 --- Over-expression of JAG2 and FZD3 Proteins in CRC Tissues --- p.72 / Chapter 3.3.3 --- FZD3 Over-expression Correlated with Tumour-Node Metastasis (TNM) stages --- p.76 / Chapter 3.3.4 --- JAG2 and FZD3 Protein Expression in Colorectal Cancer and Normal Cell Lines --- p.77 / Chapter 3.4 --- Discussions --- p.78 / Chapter Chapter 4 --- Functional Analyses of JAG2 and FZD3 in CRC Cell Lines by RNA Interference --- p.81 / Chapter 4.1 --- Background --- p.81 / Chapter 4.2 --- Materials and Methods --- p.84 / Chapter 4.2.1 --- Transfection of siRNA into CRC Cell Lines --- p.84 / Chapter 4.2.2 --- Cell Proliferation Assay --- p.85 / Chapter 4.2.3 --- Monolayer Scratch Wound Healing Assay --- p.85 / Chapter 4.2.4 --- Matrigel Invasion Assay --- p.86 / Chapter 4.2.5 --- Statistical Analysis --- p.87 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Knockdown of JAG2 and FZD3 Expression by RNA Interference --- p.88 / Chapter 4.3.2 --- Effect of JAG2 Knockdown on Migration of CRC Cell Lines --- p.91 / Chapter 4.3.3 --- JAG2 Knockdown by siRNA 2 Transfection Reduced Migratory Capability of HCT116, DLD-1and HT29 cell lines --- p.94 / Chapter 4.3.4 --- JAG2 Knockdown Impaired the Invasiveness of HCT116 and DLD-1 Cell Lines --- p.97 / Chapter 4.3.5 --- Decreased Migratory and Invasive Capabilities Induced by JAG2 Knockdown was not Due to Reduced Cell Proliferation --- p.100 / Chapter 4.4 --- Discussions --- p.102 / Chapter Chapter 5 --- NOTCH Pathway Inactivation by JAG2 Silencing Reduces Oncogenic Properties of HT29 but not HCT116 andDLD-1 CRC Cell Lines --- p.106 / Chapter 5.1 --- Background --- p.106 / Chapter 5.2 --- Materials and Methods --- p.109 / Chapter 5.2.1 --- CRC Cell lines --- p.109 / Chapter 5.2.2 --- Pharmacological Inhibition of NOTCH signaling by DAPT --- p.109 / Chapter 5.2.3 --- Combination of DAPT Treatment and JAG2 Silencing by siRNA --- p.109 / Chapter 5.2.4 --- Western Blotting --- p.109 / Chapter 5.2.5 --- Cell Proliferation Assay (MTS Assay) --- p.110 / Chapter 5.2.6 --- Monolayer Scratch Wound Healing Assay --- p.110 / Chapter 5.2.7 --- Matrigel Invasion Assay --- p.111 / Chapter 5.2.8 --- Statistical Analysis --- p.111 / Chapter 5.3 --- Results --- p.112 / Chapter 5.3.1 --- JAG2 Silencing Down-regulates Notch Pathway Signaling in CRC Cell Lines --- p.112 / Chapter 5.3.2 --- Inhibition of NOTCH Signaling by DAPT Treatment in CRC Cell Lines --- p.112 / Chapter 5.3.3 --- NOTCH Inhibition Does not Significantly Affect Cell Proliferation in CRC Cell Lines --- p.114 / Chapter 5.3.4 --- Suppression of NOTCH Signaling by DAPT Inhibits Migration in HT29 but not in HCT116 and DLD-1 CRC Cell Lines --- p.115 / Chapter 5.3.5 --- Suppression of NOTCH Signaling by DAPT does not Significantly Affect Invasiveness of HCT116 and DLD-1 CRC Cell Lines --- p.117 / Chapter 5.4 --- Discussions --- p.118 / Chapter Chapter 6 --- JAG2 Knockdown Inhibits Invasion in CRC Cell Lines through Inactivation of Cathepsin K --- p.121 / Chapter 6.1 --- Background --- p.121 / Chapter 6.2 --- Materials and Methods --- p.123 / Chapter 6.2.1 --- Human Tumour Metastasis RT2 Profiler[superscript TM] PCR Array --- p.123 / Chapter 6.2.2 --- Measurement of CTSK Gene expression level by Quantitative Real-Time PCR --- p.123 / Chapter 6.2.3 --- Immunohistochemical Staining (IS) of CTSK in CRC Tissues --- p.124 / Chapter 6.2.4 --- Pharmacological Inhibitior of CTSK in CRC Cell Lines --- p.124 / Chapter 6.2.5 --- Inhibition of CTSK in CRC Cell Lines for Migration Study --- p.124 / Chapter 6.2.6 --- Inhibition of CTSK in CRC Cell Lines for Invasion Study --- p.125 / Chapter 6.2.7 --- Western Blotting --- p.125 / Chapter 6.2.8 --- Statistical Analysis --- p.125 / Chapter 6.3 --- Results --- p.126 / Chapter 6.3.1 --- Identification of Metastasis Related Genes Which were Down-regulated by JAG2 Knockdown in HCT116 Cells --- p.126 / Chapter 6.3.2 --- Validation of Down-regulation of CTSK Gene by JAG2 Knockdown in HCT116 Cell Line by qRT-PCR --- p.126 / Chapter 6.3.3 --- JAG2 Knockdown Reduced Expression of Active CTSK Protein in CRC Cell Lines --- p.128 / Chapter 6.3.4 --- CTSK Protein Expression in CRC Tissue Samples --- p.130 / Chapter 6.3.5 --- Pharmacological Inhibition of CTSK Suppressed Invasiveness of CRC Cell Lines --- p.131 / Chapter 6.3.6 --- Pharmacological Inhibition of CTSK did not Affect Migration of CRC Cell Lines --- p.132 / Chapter 6.4 --- Discussions --- p.133 / Chapter Chapter 7 --- Depletion of JAG2 Inhibits Migration and Invasion in CRC Cell Lines through Inactivation of p38 MAPK/HSP27 Pathway --- p.137 / Chapter 7.1 --- Background --- p.137 / Chapter 7.2 --- Materials and Methods --- p.140 / Chapter 7.2.1 --- Pharmocological Inhibition of p38 MAPK Phosphorylation CRC Cell Lines --- p.140 / Chapter 7.2.2 --- Inhibition of p38 MAPK Phosphorylation for Migration Study in CRC Cell Lines --- p.140 / Chapter 7.2.3 --- Inhibition of p38 MAPK Phosphorylation for Invasion Study in CRC Cell Lines --- p.140 / Chapter 7.2.4 --- Knockdown of STAT3 by RNA interference --- p.141 / Chapter 7.2.5 --- Knockdown of STAT3 for Migration Study in CRC Cell Lines --- p.141 / Chapter 7.2.6 --- Knockdown of STAT3 for Invasion Study in CRC Cell Lines --- p.141 / Chapter 7.2.7 --- Western Blotting --- p.141 / Chapter 7.2.8 --- Statistical Analysis --- p.142 / Chapter 7.3 --- Results --- p.143 / Chapter 7.3.1 --- JAG2 Knockdown Inhibits p38 MAPK / HSP27 Pathway in CRC Cell Lines --- p.143 / Chapter 7.3.2 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Invasive Capability of CRC Cell Line --- p.145 / Chapter 7.3.3 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Migration of CRC Cell lines --- p.147 / Chapter 7.3.4 --- JAG2 Knockdown Inactivated p38 MAPK / HSP27 Pathway Independently of NOTCH Pathway in CRC Cell Lines --- p.149 / Chapter 7.3.5 --- JAG2 Knockdown Inhibits STAT3 Activation in CRC Cell Lines --- p.151 / Chapter 7.3.6 --- STAT3 Silencing Reduced Invasive Capability in CRC Cell Lines --- p.152 / Chapter 7.3.7 --- STAT3 Silencing Reduced Migratory Capability in CRC Cell Lines --- p.154 / Chapter 7.3.8 --- Inhibition of p38 MAPK Activity Suppressed STAT3 Activation in HCT116 Cells --- p.156 / Chapter 7.4 --- Discussions --- p.157 / Chapter Chapter 8 --- Conclusions and Future Works --- p.161 / Chapter 8.1 --- Conclusions --- p.161 / Chapter 8.2 --- Future work --- p.163 / References --- p.164 / Chapter Appendix 1 --- List of Figures and Tables --- p.208 / Chapter Appendix 2 --- Abbrevations used in this thesis --- p.212

published_or_final_version / Medicine / Master / Master of Medical Sciences

published_or_final_version / Diagnostic Radiology / Doctoral / Doctor of Philosophy

Colorectal cancer is second only to lung cancer as a leading cause of internal cancer death. Individuals over 65 years of age are most at risk yet least likely to engage in screening for colorectal cancer. The purpose of this descriptive-correlational study using a modified Pender Health Promotion Model was to identify motivations of elderly individuals to engage in health protective behavior. A convenience sample of 90 subjects answered a four-part motivations questionnaire in which three subscales--early detection, powerful others, and chance--met reliability standards (alpha >.70). Chance was significantly related to compliance (r = -.28; p =.003); Hemoccult compliers believed less in chance and powerful others than did non-compliers (p =.005;.002). The 88 percent who performed a Hemoccult stool test as a screening method for early detection of colorectal cancer demonstrated that these elders willingly engaged in health protective behavior and supported the nurses' role in promoting primary prevention in elderly clients.

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

Laparoscopic surgery for colorectal cancer was first reported in 1991. However, early experiences with laparoscopic colectomy were unfavorable, with higher than expected rates of port-site recurrence and concerns about compromised long-term oncologic outcomes. These concerns have been resolved by the results of several large-scale European and American multicenter randomized controlled trials (RCTs) that reported no difference in oncologic clearance and survival between laparoscopic and open colectomy for colon cancer. / The role of laparoscopic surgery for rectal cancer, on the other hand, still remains controversial. Because laparoscopic surgery for rectal cancer is technically more difficult and has a higher morbidity rate than laparoscopic colectomy for colon cancer, most of the published large-scale multicenter RCTs comparing laparoscopic and open colorectal cancer did not include patients with rectal cancer. To date, good-quality data comparing laparoscopic and open surgery for rectal cancer are still scarce in the literature. The main objective of this thesis is to provide additional evidence to justify the role of laparoscopic surgery for rectal cancer. / To be justified, laparoscopic surgery for rectal cancer should have equal or better clinical outcomes than open surgery and improve quality of life. Furthermore, oncologic clearance as well as long-term survival should not be adversely affected by the laparoscopic approach. / In this thesis, a series of RCTs and comparative studies with long-term follow-up were conducted to address the above issues. Our results demonstrate that laparoscopic surgery for rectal cancer is associated with earlier postoperative recovery, better preservation of urosexual function and quality of life, and less late morbidity when compared with open surgery. Oncologic clearance in terms of resection margins and number of lymph nodes harvested are comparable between the laparoscopic and open groups. Most importantly, laparoscopic surgery does not adversely affect disease control or jeopardize long-term survival of rectal cancer patients. The benefits of the laparoscopic over the open approach remain the same regardless of the types of rectal cancer surgery (laparoscopic-assisted anterior resection, total mesorectal excision, or abdominoperineal resection) or the location of the tumor. It is therefore concluded that laparoscopic surgery for rectal cancer is safe and justified. Based on our results, we believe that laparoscopic surgery can be regarded as an acceptable alternative to open surgery for treating curable rectal cancer. / Ng, Siu Man Simon. / Thesis (M.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 323-366). / Dedication --- p.1 / Declaration of Originality --- p.2 / Abstract --- p.3 / Table of Contents --- p.5 / List of Tables --- p.8 / List of Figures --- p.10 / List of Abbreviations --- p.13 / PRÉCIS TO THE THESIS --- p.15 / Chapter PART I --- BACKGROUND --- p.37 / Chapter Chapter 1 --- Management of Colorectal Cancer: From Open to Laparoscopic Surgery --- p.38 / Chapter 1.1 --- Introduction to Colorectal Cancer --- p.39 / Chapter 1.2 --- A Brief History of Laparoscopic Surgery --- p.51 / Chapter 1.3 --- Laparoscopic Colorectal Surgery: The Beginning --- p.58 / Chapter 1.4 --- Evidence for the Safety and Efficacy of Laparoscopic Surgery for Colon Cancer --- p.62 / Chapter Chapter 2 --- Laparoscopic Surgery for Rectal Cancer: A Critical Appraisal of Published Literature --- p.71 / Chapter 2.1 --- Introduction --- p.72 / Chapter 2.2 --- Evidence from Single-Center Trials --- p.76 / Chapter 2.3 --- Evidence from Multicenter Trials --- p.82 / Chapter 2.4 --- Ongoing Trials --- p.89 / Chapter 2.5 --- Discussion --- p.92 / Chapter Chapter 3 --- Laparoscopic Surgery for Rectosigmoid and Rectal Cancer: Experience at The Prince of Wales Hospital, Hong Kong --- p.97 / Chapter 3.1 --- The Beginning of Laparoscopic Era in Hong Kong --- p.98 / Chapter 3.2 --- Early Experience of Laparoscopic Colorectal Surgery --- p.102 / Chapter 3.3 --- Nonrandomized Comparative Studies --- p.105 / Chapter 3.4 --- The Hong Kong Trial --- p.110 / Chapter PART II --- HYPOTHESES AND CLINICAL STUDIES --- p.116 / Chapter Chapter 4 --- Research Hypotheses and Objectives --- p.117 / Chapter 4.1 --- Research Hypotheses --- p.118 / Chapter 4.2 --- Research Plan and Objectives --- p.120 / Chapter Chapter 5 --- Laparoscopic-Assisted Versus Open Anterior Resection for Upper Rectal Cancer: Short-Term Outcomes --- p.122 / Chapter 5.1 --- Abstract --- p.123 / Chapter 5.2 --- Introduction --- p.125 / Chapter 5.3 --- Patients and Methods --- p.128 / Chapter 5.4 --- Results --- p.133 / Chapter 5.5 --- Discussion --- p.144 / Chapter 5.6 --- Conclusions --- p.148 / Chapter Chapter 6 --- Laparoscopic-Assisted Versus Open Anterior Resection for Upper Rectal Cancer: Long-Term Morbidity and Oncologic Outcomes --- p.149 / Chapter 6.1 --- Abstract --- p.150 / Chapter 6.2 --- Introduction --- p.152 / Chapter 6.3 --- Patients and Methods --- p.154 / Chapter 6.4 --- Results --- p.158 / Chapter 6.5 --- Discussion --- p.173 / Chapter 6.6 --- Conclusions --- p.179 / Chapter Chapter 7 --- Laparoscopic-Assisted Versus Open Abdominoperineal Resection for Low Rectal Cancer --- p.180 / Chapter 7.1 --- Abstract --- p.181 / Chapter 7.2 --- Introduction --- p.183 / Chapter 7.3 --- Patients and Methods --- p.185 / Chapter 7.4 --- Results --- p.190 / Chapter 7.5 --- Discussion --- p.201 / Chapter 7.6 --- Conclusions --- p.207 / Chapter Chapter 8 --- Laparoscopic-Assisted Versus Open Total Mesorectal Excision with Anal Sphincter Preservation for Mid and Low Rectal Cancer --- p.208 / Chapter 8.1 --- Abstract --- p.209 / Chapter 8.2 --- Introduction --- p.211 / Chapter 8.3 --- Patients and Methods --- p.214 / Chapter 8.4 --- Results --- p.221 / Chapter 8.5 --- Discussion --- p.238 / Chapter 8.6 --- Conclusions --- p.246 / Chapter Chapter 9 --- Long-Term Oncologic Outcomes of Laparoscopic Versus Open Surgery for Rectal Cancer: A Pooled Analysis of Three Randomized Controlled Trials --- p.247 / Chapter 9.1 --- Abstract --- p.248 / Chapter 9.2 --- Introduction --- p.250 / Chapter 9.3 --- Patients and Methods --- p.254 / Chapter 9.4 --- Results --- p.258 / Chapter 9.5 --- Discussion --- p.272 / Chapter 9.6 --- Conclusions --- p.280 / Chapter Chapter 10 --- Prospective Comparison of Quality of Life Outcomes After Curative Laparoscopic Versus Open Sphincter-Preserving Resection for Rectal Cancer --- p.281 / Chapter 10.1 --- Abstract --- p.282 / Chapter 10.2 --- Introduction --- p.284 / Chapter 10.3 --- Patients and Methods --- p.287 / Chapter 10.4 --- Results --- p.292 / Chapter 10.5 --- Discussion --- p.308 / Chapter Chapter 11 --- Conclusions --- p.314 / Chapter 11.1 --- Conclusions --- p.315 / REFERENCES --- p.322 / LIST OF PUBLICATIONS RELATED TO THE THESIS --- p.367 / ACKNOWLEDGEMENTS --- p.373

Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7094. Adviser: Yun-Xin Fu. Includes bibliographical references.

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Medical Sciences