O câncer colorretal (CCR) é uma das maiores causas de óbito no mundo industrializado, com uma incidência anual de 800.000 casos novos, o que significa 8,5% de todos os novos e 12% das mortes relacionadas a essa doença. No Brasil, excluindo-se os cânceres de pele não melanoma, o CCR é o quarto mais freqüente entre os homens e o terceiro entre as mulheres. O risco de desenvolver CCR é de aproximadamente 5% a 6% na população ocidental. Existem evidências epidemiológicas de redução do CCR em 60% -90% quando a colonoscopia com polipectomia é usada preventivamente A colonoscopia ainda é o melhor método para o diagnóstico precoce do CCR e das lesões precursoras. Contudo existem falhas de detecção não desprezíveis. O objetivo deste estudo foi comparar o resultado do exame detalhado da mucosa do cólon e do reto através da colonoscopia convencional, da cromoendoscopia e do NBI, na detecção de lesões elevadas, deprimidas e planas em pacientes submetidos ao exame sem antecedentes pessoais e ou familiares. Entre janeiro de 2007 e outubro de 2009 foram selecionados 181 pacientes divididos aleatoriamente em três grupos: A: 48 pacientes, controle; B: 29 pacientes, NBI; C: 104 pacientes, cromoscopia difusa. Pode-se observar que dos 181 pacientes examinados 38 (21%) não apresentavam lesões. Os 143 pacientes com lesão, apresentaram um número médio de 2,65 lesões, com mínimo de 1 e máximo de 7 lesões. Nos total dos 181 pacientes e no conjunto dos 143 pacientes com lesões não foi observada diferença estatisticamente significante entre os três grupos A, B e C para a idade, o tempo reto-ceco e o tempo ceco-reto, enquanto que para a altura, peso e conseqüente IMC houve variação estatística. O tamanho médio das 379 lesões encontradas nos 143 pacientes, avaliado pelo seu diâmetro foi de 5,45 ± 2,84 mm, sem variação estatística entre os grupos, entre os hemicólons e entre os hemicólons nos grupos. Os tamanhos das lesões foram reunidos em três intervalos distintos: até 5 mm (76,30%), de 6 a 10 mm (19,50%) e de 11 a 20 mm (4,20%). Do total de 379 lesões, 203 (53,6%) mostraram-se neoplásicas e 176 (46,4%) não neoplásicas. O tamanho médio das 203 lesões neoplásicas foi de 5,96 mm, e das 176 não neoplásicas, 4,87 mm. As lesões neoplásicas mostraram-se maiores que as não neoplásicas, com significância estatística. Nos grupos não houve variação significante entre neoplasia e não neoplasia, mas diferença significante entre o tamanho das neoplasias e não neoplasias. Não houve diferença estatística entre os tamanhos das lesões nos dois hemicólons, mas com diferença significante entre os tamanhos das lesões neoplásicas e não neoplásicas. O mesmo se observa quando os segmentos do cólon são analisados individualmente. Os dois segmentos que apresentaram diferença significante, especificamente, quanto ao tamanho das lesões neoplásicas e não neoplásicos foram o sigmóide e o transverso. Nota-se que todas as lesões subpediculadas e as lesões plano-elevadas com depressão central eram neoplásicas. As lesões planas e neoplásicas são proporcionalmente mais visíveis no hemicólon direito nos grupos B (85,7%) e C (67,9%), sem diferença estatística. As hipóteses diagnósticas das lesões feitas durante o exame colonoscópico foram comparadas com os resultados histopatológicos. Pode-se observar que no grupo A sensibilidade de 82,7%, especificidade de 59%, com taxa de concordância de 72,5 %, considerada regular, no grupo B sensibilidade de 92,3%, especificidade de 61,9%, com concordância de 78,7 %, regular e no C sensibilidade de 88,8%, especificidade de 79,3%, taxa de concordância de 84,2%, considerada boa. Proporcionalmente o grupo C tem maior número de pacientes com três ou mais lesões e três ou mais lesões neoplásicas, mas sem valor estatístico. Conclui-se que não houve diferença estatística entre os 181 pacientes examinados e os 143 pacientes com lesões, quanto aos dados gerais, não houve diferenças significativas quanto ao número relativo, ao tipo e ao tamanho das lesões. As lesões neoplásicas apresentam-se maiores quando comparadas às não-neoplásicas, com significância estatística. A concordância entre a hipótese diagnóstica colonoscópica e a histologia é maior no grupo da cromoscopia / Colorectal cancer (CRC) is one of the largest causes of death on the industrialized world. Its annual incidence of 800.000 new cases means 8,5% of all the new ones and 12% of deaths related to this disease. In Brazil, excluding the non-melanoma skin-cancers, CRC is the fourth more frequent among men and the third one among women. The risk for developping CRC is approximately of 5 to 6% on the Western population. There are epidemiological evidences for reducing CRC on 60-90% when colonoscopy with polypectomy is used preventively. Colonoscopy is still the best method both for the early dyagnosis of CRC and precursor lesions. However, there are non-contemptible failures on the detection. This paper purpose was comparing the result of colon and rectum mucous membrane detailed test through conventional colonoscopy, chromoendoscopy and NBI, on the detection of augmented, depressed and flat lesions in patients submitted to it without any personal or familiar antecedents. Between January 2007 and October 2009 181 patients were selected randomically and divided into 3 groups: A: 48 control patients; B: 29 patients, NBI; C: 104 patients, diffuse chromoscopy. It is observed that, from the 181 examined patients, 38 (21%) didnt present lesions. The 143 patients with lesion, presented an average number of 2,65 lesions, with a minimum of 1 and a maximum of 7 lesions. On the total of the 181 patients and on the whole of the 143 patients with lesions it was not observed any statistically significant difference among the three groups A, B and C as for Age, the Rectum-Cecum Time and the Cecum-Rectum Time, while there was a statistical variation for Height, Weight and consequent bmi. The average size of the 379 lesions found on the 143 patients, assessed by its diameter was of 5,45 mm (2.14 in.) + 2,84 mm (1,11 in), without any statistical variation among the groups, among the hemicolons and among the hemicolons in the groups. The size of the lesions were gathered into three distinct intervals: up to 5 mm [1.9 in.] (76,30%), from 6 mm [2.3 in.] to 10 mm [3.9 in] (19,50%) and from 11 to 20 mm [4.3 to 7.8 in] (4,20%). From the total of 379 lesions, 203 (53,6%) revealed themselves neoplastic and 176 (46,4%) non-neoplastic. The average size of the 203 neoplastic lesions was of 5,96 mm (2.34 in.), and of the 176 non-neoplastic ones, 4,87 mm [12,36 in]. Neoplastic lesions have shown larger than the non-neoplastic ones, with a stastistical significance. On the groups there is any significant variation between neoplasia and non-neoplasia, but a significant difference between the neoplasias and non-neoplasias size. There was any statistical difference among the lesion size on both hemicolons, however, a significant difference among the sizes of neoplastic and non-neoplastic ones. The same is observed when colon segments were analyzed individually. The two segments that have presented significant lesions, specifically on what concerns the size of neoplastic and non-neoplastic ones were the Sigmoid and the Transverse. It is observed that all the subpediculated lesions and the flat-augmented ones with a central depression were neoplastics. The flat and neoplastic lesions are proportionally more visible on the right hemicolon at groups B (85,7%) and C (67,9%), without any statistical difference. The diagnostic hypotheses of the lesions grown during the colonoscopic test were compared to the histopathological results. On control group (A) it is observed a 82,7% sensibility, a 59% specificity with a concordance rate of 72,5%, considered regular. On group B it is observed a 92,3% sensibility, a 61,9% specificity, with a regular concordance rate of 78,7%. On group C it is observed a 88,8% sensibility, a 79,3% specificity, a 84,2% concordance rate, considered good. Proportionally group C has a larger number of patients with 3 or more lesions and or more neoplastic lesions, but with no statistical value. On what concerns general data, it is concluded that there wasnt any statistical difference among the 181 patients examined and the 143 ones presenting lesions as for the relative number, the type and size of the lesions. Neoplastic lesions appear to be larger when compared to non-neoplastic ones, with a statistical significance. The concordance between the colonoscopic diagnostic hypothesis and the histology is larger on chromoscopy group

INTRODUÇAO: O câncer de cólon é a terceira neoplasia mais prevalente no país, com aumento progressivo da incidência associada ao envelhecimento populacional. Os avanços nos tratamentos local e sistêmico do câncer de cólon metastático tem aumentado significativamente o tempo de sobrevida global. Entretanto, ainda não existem biomarcadores consolidados na literatura, capazes de predizer resposta a estes tratamentos ou o prognóstico. No processo da carcinogênese, uma das importantes vias que se encontra alterada é a via de reparo do DNA. A Chk2 é uma proteína quinase com atividade no reparo celular atuando de forma supressora no processo da carcinogênese, sendo que alterações em sua expressão e/ou função têm sido associadas à progressão tumoral em outras neoplasias como no câncer de mama, pulmão, vulva, bexiga, cólon, ovário, osteossarcoma e linfomas. OBJETIVO: Avaliar a expressão imunohistoquímica do Chk2 no câncer de cólon metastático e correlacionar sua expressão com características clínico-patológicas e sobrevida. PACIENTES E MÉTODOS: Foram incluídos 58 pacientes com diagnóstico confirmado de câncer de cólon metastático, tratados em primeira linha com quimioterapia baseada em fluorouracila e oxaliplatina. O tempo mínimo de seguimento foram de 2 anos pós-diagnóstico. Para análise da expressão do Chk2 foram utilizadas as técnicas de tissue microarray e imunohistoquímica. Estes resultados foram correlacionados com características clínicas, patológicas e de sobrevida. Para análise estatística, foi utilizado o programa SPSS17 e o valor de p<0,05 foi considerado estatisticamente significativo. RESULTADOS: A expressão de Chk2 foi positiva em 69% dos pacientes. Houve associação entre a expressão de Chk2 e o status linfonodal (p = 0,012) e entre a sobrevivência (p=0,034). A expressão negativa de Chk2 aumentaram as chances de envolvimento linfonodal (OR:10,2, p=0,03). O tempo de sobrevida global de pacientes Chk2 negativo foi maior (72 versus 59 meses, p=0,155), o mesmo foi observado com o tempo sobrevida livre de progressão (19 versus 13 meses, p=0,293). As curvas de sobrevida foram diferentes de acordo com a expressão do Chk2 em pacientes com ou sem envolvimento linfonodal, sendo menor nos pacientes com Chk2 positivo, p=0,028. Houveram mais óbitos em pacientes com Chk2 positivo. Análise multivariada identificou o performance status segundo a escala de ECOG (p=0,001 ); metástase sincrônica (p=0,037); diferenciação das células tumorais (p=0,029) e expressão de Chk2 (p=0,020) como fatores independentes para sobrevida global. CONCLUSÃO: A expressão positiva do Chk2 no adenocarcinoma de cólon metastático foi indicativa de maior agressividade e disseminação tumoral, impactando de forma negativa na sobrevida e desfecho dos pacientes. / INTRODUCTION: The DNA damage checkpoint pathway has been of interest to the field of cancer biology, since checkpoint defects result in the accumulation of altered genetic information, a central feature of carcinogenesis. Little is known about the role of Chk2 in colorectal cancer tumorigenesis. OBJECTIVE: The purpose of this study was to evaluate Chk2 expression in metastatic colon cancer and correlate this with clinicopathological features and patient survival. PATIENTS AND METHODS: Tissues were obtained from 58 patients with confirmed metastatic colon cancer diagnosis, treated with capecitabine and oxaliplatin chemotherapy as standard doses. Patients included had, at least, 2 years post diagnosis of clinical following. The tissue microarray immunohistochemistry was the technic to evaluate Chk2 expression. Statistics analysis used SPSS 17. A p-value <0,050 was considered to be statistically significant. Immunohistochemical expression of Chk2 and its relationship with clinical and pathological characteristics and survival data was reported. RESULTS: The expression of Chk2 was positive in 69%. There was association between expression of Chk2 and Iymph node status (p=0.012) and between survival (p=0.034). The negative expression of Chk2 enhanced the chances of linfonodal involvement (OR:10,2, p=0.03). The global survival time of Chk2 negative patients was higher (72 versus 59 months, p= 0.155); the same was observed with progression-free survival time (19 versus 13 months, p=0.293). The survival curves were different according to Chk2 expression in patients with or without Iymph node involvement, being lower in patients with Chk2 positive, p=0.028. There were more deaths in patients with Chk2 positive. Multivariate regression analysis identified performance status ECOG (p=0.001), synchronous metastasis (p=0.037), tumor cell differentiation (p=0.029) and expression of Chk2 (p=0.020) as independent factors to overall survival. CONCLUSION: This study demonstrated that the Chk2 positive expression in colon cancer indicates increased tumor spread and tumoral aggressiveness, impacting negatively on survival and outcome of patients.

Le sperme est le principal vecteur de dissémination du VIH. La muqueuse colorectale exposée au sperme infecté lors de rapports anaux, aussi bien chez l’homme que chez la femme, représente le plus fort risque d’infection parmi toutes les voies de transmission sexuelle. Un nombre croissant d’études suggèrent que la transmission sexuelle du VIH via les cellules infectées présentes dans les sécrétions génitales du donneur serait plus efficace que par les particules virales libres. A ce jour, le rôle et les mécanismes de transmission des cellules infectées du sperme ont été peu étudiés. De plus, l’effet potentiel du liquide séminal (LS) sur cette transmission est mal connu. Dans ce contexte, mes travaux de thèse ont permis de démontrer une transmigration active et rapide: (i) de leucocytes sanguins à travers la barrière colorectale, et un effet inhibiteur du LS sur cephénomène, sans altération de la barrière épithéliale. (ii) de leucocytes séminaux. Des analyses par cytométrie de flux ont permis de mettre évidence à la fois des particularités et des similitudes quant à l’équipement protéique des cellules séminales et de leurs équivalents sanguins. Ce travail fournit de nouvelles données sur la transmission du VIH par les cellules infectées du sperme, et sur l’effet du sperme sur la transmission colorectale. La démonstration de la transmigration de leucocytes séminaux suggère que ces cellules jouent un rôle dans la transmission du VIH au niveau colorectale qui nécessite d’être pris en compte dans les stratégies de prévention. Les mécanismes en jeu et ceux responsables de l’effet du LS restent à élucider et devrait permettre à terme de dégager de nouvelles cibles thérapeutiques. / Semen is the main vector of HIV dissemination. The colorectal mucosa exposed to semen infected cells during anal intercourse, in both men and women, represents the highest risk of infection among all sexual transmission routes. An increasing number of studies suggest that sexual transmission of HIV via infected cells present in the donor's genital secretions would be more effective than free viral particles. To date, the role and transmission mechanisms of infected sperm cells have been poorly studied. Moreover, the potential effect of seminal fluid (SF) on this transmission is poorly understood. In this context, my thesis work has demonstrated an active and rapid transmigration: (i) of blood leukocytes across the colorectal barrier, and an inhibitory effect of SF on this phenomenon, without altering the epithelial barrier. (ii) of seminal leucocytes. Flow cytometry analyzes have revealed both features and similarities in the protein equipment of seminal cells and their blood equivalents. This work provides new data on HIV transmission by infected sperm cells, and on the effect of semen on colorectal transmission. Demonstration of transmigration of seminal leukocytes suggests that these cells play a rôle in colorectal HIV transmission that needs to be considered in prevention strategies. The mechanisms involved and those responsible for the effect of SF remain to be elucidated and should eventually lead to new therapeutic targets.

O presente estudo teve como objetivo investigar se existe uma correlação entre ansiedade, depressão e níveis séricos de citocinas de pacientes com câncer colorretal em diferentes estágios da terapia antitumoral. Para tanto, utilizamos um total de 100 indivíduos, divididos em cinco grupos amostrais. Grupo 1. Voluntários saudáveis livres de qualquer doença psiquiátrica ou associada com alterações do sistema imune (n=20); Grupo 2. Pacientes com diagnóstico de adenocarcinoma colorretal confirmado e que não foram submetidos à ressecção cirúrgica (n=20); Grupo 3. Pacientes submetidos à ressecção cirúrgica e que não iniciaram a terapia adjuvante (n=20); Grupo 4. Pacientes em tratamento quimioterápico há cerca de 3 meses, independentemente do esquema de quimioterapia aplicado (n=20); Grupo 5. Pacientes que concluíram o esquema de quimioterapia adjuvante há cerca de 6 meses (n=20). Depressão e ansiedade foram analisados utilizando Hospital Anxiety and Depression Scale (HADS) e níveis séricos de IL-1, IL-6, IL-8, IL-10, IL-12, TNF-?, TGF-? e Fractalcina foram mensurados por CBA. Níveis clinicamente relevantes de ansiedade e/ou depressão foram verificados em todos os pacientes com CCR em diferentes estágios da terapia antitumoral. Um padrão de produção semelhante foi observado para as citocinas pró-inflamatórias avaliadas. IL-1, IL-6, IL-8, TNF-? e Fractalcina foram encontradas em níveis elevados nos pacientes com CCR em estágio pré (G2) e pós-operatório (G3). Estes níveis foram, contudo, reduzidos durante (G4) e após (G5) o tratamento quimioterápico. Além disso, verificamos níveis diminuidos de IL-10 no soro dos pacientes dos grupos 2 e 3 (pré e pós-operatório). Ao analisarmos a correlação entre a pontuação HADS e os níveis séricos de citocinas, observamos uma associação positiva de ansiedade e/ou depressão com as concentrações de IL-1, IL-6, IL-8, TNF-? e Fractalcina e negativa com IL-10 em pacientes nos diferentes estágios da terapia antitumoral. Estes resultados indicam haver uma importante correlação entre os níveis séricos de citocinas pró-inflamatórias, ansiedade e depressão em pacientes com câncer colorretal, sugerindo que tais citocinas estão envolvidas na patofisiologia dessas comorbidades / This study aimed to investigate whether there is a correlation between anxiety, depression and serum cytokine levels in colorectal cancer in different stages of the antitumor therapy. A sample of 100 subjects was divided in 5 groups. Group 1: Healthy volunteers free of any psychiatric or immune system disease (n=20); Group 2: Patients with colorectal cancer who did not undergo surgical resection (n=20); Group 3: Patients who underwent surgical resection and who did not start adjuvant therapy (n=20); Group 4: Patients undergoing chemotherapy for about 3 months, regardless of chemotherapy protocols applied (n=20); Group 5: Patients who have completed adjuvant chemotherapy regimen for about 6 months (n=20). Depression and anxiety were analyzed using the Hospital Anxiety and Depression Scale (HADS), and serum levels of IL-1, IL-6, IL-8, IL-10, IL-12, TNF-?, TGF-?, and Fractalkine were measured by CBA. Clinically relevant levels of anxiety and/or depression were found in all CRC patients in different stages of the antitumor therapy. A similar pattern of production was observed for proinflammatory cytokines. Elevated levels of IL-1, IL-6, IL- 8, TNF-?, and Fractalkine were found in CRC patients in pre (G2) and postoperative (G3) stages. However, these levels were reduced during (G4) and after (G5) chemotherapy. Furthermore, we found decreased levels of IL-10 in serum of patients in CRC patients in pre and postoperative stages. By analyzing the correlation between HADS scores and serum cytokine levels, we observed a positive association of anxiety and/or depression with the concentrations of IL-1, IL-6, IL-8, TNF-?, and Fractalkine, and negative with IL-10 in patients in different stages of the antitumor therapy. These results indicate an important link between serum levels of proinflammatory cytokines, anxiety and depression in CRC patients, suggesting that such cytokines are involved in the pathophysiology of these comorbidities

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

Inflammatory bowel disease (IBD) confers a high risk of development of colitis-associated colorectal cancer in patients with extensive colitis. Crypt fission is a mechanism of clonal expansion in the intestinal epithelium. Although fission is rare in the normal colon, many crypts in IBD patients are in the process of fission. Protumourigenic mutations can spread through the entire inflamed colon relatively quickly indicating that stem cell dynamics are altered in IBD. Some patients with IBD develop pseudopolyps as a result of mucosal ulceration and epithelial regeneration. The aim of this PhD was to investigate the effect of inflammation on niche succession, the crypt cycle and the expansion of clones in the IBD intestine. Pseudopolyps were examined as potential sites for clonal expansion by determining the frequency of mutated pseudopolyps and proliferative potential, and examining their microRNA (miRNA) profile relative to inactive, active and dysplastic mucosa, and adenoma and cancerous tissue. This thesis will show that crypt fission cycles in inflammatory bowel diseased colon are protracted and that each stage of crypt fission appears to be slow. Overall, clonally related adjacent IBD crypts seem to share a more recent common ancestor than non-related IBD crypts, supporting increased crypt fission rates in IBD. The proliferative drive induced by continuous inflammation and mucosal repair in ulcerative colitis (UC) appears to promote the expansion of CCO-deficient patches. Furthermore, niche succession appears to be faster in active IBD. Pseudopolyps are a source of regeneration within the epithelium and, as shown here, have a faster proliferative drive than background mucosa in IBD patients. Pseudopolyps are not genetically inert and are a potential source of protumourigenic mutations in UC. Hence, pseudopolyps are a potential reservoir within the inflamed epithelium where mutations are harboured and where there is no competition from neighbouring epithelium, as it has been denuded following previous inflammation. MiRNA expression in pseudopolyps differs from that of UC-dysplasia and mucosa. In particular, the MiR-29 family was downregulated in pseudopolyps, a miRNA family that has been implicated in intestinal fibrosis formation in stricturing Crohn’s disease. Pseudopolyps have been traditionally thought of as benign, genetically inert and incidental findings characteristic of chronic inflammation. My research runs counter to this view indicating an exciting paradigm shift in the way we consider pseudopolyps, which may eventually alter the endoscopic management of these lesions in the future.

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

Mutations in the tumour suppressor gene Adenomatous polyposis coli (APC) are found in 80 % of sporadic colorectal cancer (CRC) tumours and are also responsible for the inherited form of CRC, Familial adenomatous polyposis (FAP). In order to identify novel therapeutic targets for the treatment of APC mutated CRC, we have generated an in vitro model of APC mutant CRC using CRISPR-cas9 gene editing. Using the APC wildtype colorectal carcinoma cell line RKO, we targeted the cells with guide RNA (gRNA) targeting exon 2 or exon 15 (encodes 80 % of APC) of the APC gene. We generated isogenic cell lines which differed in the expression of APC, the controls were APC wildtype and the APC mutant (APC Lys736fs) cell lines expressed a truncated ~80 kDa APC protein. We used these cell lines to perform an siRNA screen against 720 kinases and kinase-related genes. We selected seven genes to investigate further, unfortunately none of the potential hits validated. Additionally, we performed an FDA-approved compound screen targeting over 1000 compounds. From this, we identified a group of HMG-CoA reductase (HMGCR) inhibitors known as statins, which selectively cause a greater loss in cell viability in the APC mutated cell lines, compared to the APC wildtype cells. Mechanistically, our data suggests this synthetic lethal relationship is due to a greater decrease in the anti-apoptotic protein survivin. We propose this is due to statins altering the localisation of Rac1, reducing Pak1 activation and reducing the level of Wnt signalling. This results in the reduction of the Wnt target gene survivin. We have successfully identified an FDA-approved family of compounds, which show synthetic lethality with the APC mutation in our in vitro model.

With 50,310 related deaths this year, colorectal cancer (CRC) has emerged as the second largest cause of cancer related deaths among Americans. While 70 million Americans are considered at-risk of developing CRC, it is highly curable if detected early. Cohesin proteins, which hold sister chromatids together during replication, have emerged as a potential biomarker in multiple cancer lines. Because of their probable role in DNA replication, DNA repair, chromatin nano-architecture, and gene expression, this paper assessed whether cohesion proteins could be used as a potential biomarker for colorectal cancer risk stratification. While cohesin protein mutations have been reported in different cancers and involved in chromosomal instability, its role in early cancer formation has yet to be observed. Using immunohistochemical and Quantitative Real Time PCR analysis, this thesis assessed the protein and RNA expression levels of cohesin proteins SA-1, NIPBL, and SMC3 from human biopsies at different stages and locations of colorectal cancer development. The results showed that SA-1, a structural cohesion subunit, was significantly (p<0.01) down regulated in cancerous compared to normal tissue. The SA-1 protein was also down regulated in the involved mucosa adjacent to CRC polyps. The cohesion loading protein, NIPBL, was also significantly (p<0.01) under expressed in cancerous versus normal tissue. The RNA expression analysis of rectal mucosa showed that SMC3 and SA-1 was over expressed two fold in patients harboring hyperplastic and adenomous polyps, giving evidence that cohesin proteins are differentially expressed throughout the field of carcinogenesis. Our results demonstrate for the first time that cohesion dysregulation is an early event in human colorectal cancer development and may serve as an important biomarker of field carcinogenesis.