Multiple myeloma (MM) is an incurable haematological malignancy. It is characterized clinically by an asymptomatic precursor stage, known as monoclonal gammopathy of undetermined significance (MGUS), which will transform into symptomatic MM at a rate of 1% per year. Gene promoter hypermethylation by catalytic conversion of cytosine into 5?methylcytosine at promoter?associated CpG island is an alternative mechanism of gene inactivation. MicroRNA (miRNA) is a class of short, single?stranded, non?coding RNA molecules, which will repress the expression of target protein by sequence?specific binding to the three prime untranslated region of the corresponding messenger RNA. In carcinogenesis, miRNA can be either oncogenic when tumour suppressor genes are targeted, or tumour suppressive when oncogenes are targeted. Despite reports of hypermethylation of multiple protein?coding tumour suppressor genes, little is known about DNA methylation of non?coding tumour suppressor miRNA genes in MM. This thesis aimed to investigate the role of promoter hypermethylation of tumour suppressor miRNA genes in MM using a candidate miRNA approach. Moreover, the prognostic significance of tumour suppressor miRNA hypermethylation was studied in a uniformly?treated cohort of MM patients. The role of DNA methylation at the promoter of miR?203, miR?34a, miR?34b/c, miR?124?1, miR?129?2 and miR?224 were studied in MM. The tumour suppressor role of miR?34b/c, miR?124?1, miR?203 and miR?224 were demonstrated in human myeloma cell lines (HMCLs). In particular, restoration of miR?203 in MM cells was shown to inhibit cellular proliferation via targeting and hence direct downregulation of a proto?oncogene, cyclic AMP responsive element binding protein. There are several observations in primary MM samples. First, there was frequent methylation of miR?129?2, miR?203 and miR?224 but infrequent methylation of miR?34a, miR?34b/c and miR?124?1 in MM at diagnosis. Second, tumour?specific hypermethylation of each of the miR?203 and miR?224 promoters was detected at comparable frequencies in MGUS, diagnostic and relapsed/progressed MM, and hence implicated as an early event in myelomagenesis. Thirdly, miR?129?2 methylation was more frequent in diagnostic MM than MGUS, and hence implicated in MGUS progression to MM. On the other hand, despite rare miR?34b/c methylation at diagnosis, miR?34b/c methylation was frequent at relapse/progression, thereby implicating miR?34b/c methylation in MM relapse/progression. Fourthly, despite frequent miR?124?1 methylation in HMCLs, miR?124?1 methylation was rare in both diagnostic and relapsed MM marrow samples, suggesting that miR?124?1 methylation was acquired during in vitro cell culture. Finally, the prognostic significance of methylation of a panel of tumour suppressor miRNAs was studied in a uniformly?treated cohort of MM patients, which revealed that miR?224 hypermethylation as an independent favourable prognostic factor for survival. In conclusion, hypermethylation of tumour suppressor miRNAs is implicated in the pathogenesis (miR?203, miR?129?2, miR?224), progression (miR?34b/c), and prognostification (miR?224) of MM. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

MicroRNAs are small regulatory RNAs that bind to the seeding regions within the 3’-untranslated region (3’-UTR) of their target transcripts to modulate transcript stability and/or inhibit protein translation. MicroRNA Let-7a belonging to the Lethal-7 (Let-7) family is down-regulated at the blastocyst stage, suggesting its suppression is crucial for embryo implantation. Yet, the underlying mechanism on how Let-7a modulates blastocyst implantation remains largely unknown. In silico analysis identified attachment related integrin beta-3, outgrowth related vav3, and the microRNA processing dicer, as Let-7a targets. Therefore, it is hypothesized that down-regulation of Let-7a promotes embryo implantation by stimulating these target proteins. Let-7a is down-regulated during blastulation and at 3-hour post-estradiol activation of the dormant blastocysts. Force-expression of Let-7a in mouse blastocysts suppressed blastocyst attachment, outgrowth on fibronectin-coated plates and compromised pregnancy in vivo. Dual luciferase assay using the 3’-UTR reporter constructs of the integrin beta-3, vav3, and dicer confirmed the interaction between Let-7a and the three targets. Force-expressing or inhibiting Let-7a expression in mouse blastocysts by electroporating the Let-7a precursor or inhibitor respectively illustrated post-transcriptional regulation of Let-7a on integrin beta-3 and vav3, and transcriptional regulation on dicer. Dormant blastocysts retrieved from the delayed implanting mice expressed high Let-7a levels, which was suppressed in the first 12-hours of estradiol activation. Concomitantly, dormant blastocysts expressed low levels of integrin beta-3, vav3, and dicer, yet, their protein expression was up-regulated from 3 hours-post estradiol activation. Furthermore, addition of integrin beta-3 antibody suppressed attachment of trophoblast spheroids (blastocyst surrogate) onto endometrial epithelial cells in a co-culture model and the outgrowth of the spheroids on fibronectin-coated plates. Knockdown of Vav3 with siRNA decreased blastulation, hatching, and outgrowth rates of the embryos in vitro. Although the loss of vav3 activities did not affect embryo implantation, it suppressed trophoblast migration on fibronectin-coated plates and invasion into collagen matrix. In contrast, force-expression of vav3 enhanced blastocyst outgrowth, and promoted cell proliferation. Blocking integrin beta-3 activities in the vav3 knock-down embryos further suppressed blastocyst outgrowth, suggesting the intertwining effect of the integrins and vav3. Dicer knock-down in mouse blastocysts decreased mature Let-7a expression and suppressed blastulation and hatching in vitro and implantation in vivo. Dicer knock-down in estradiol activated mouse blastocysts decreased the epidermal growth factor receptor expression and lowered the affinity of the embryos to EGF, and suppressed the implantation potential to a level similar to that of dormant blastocysts. Taken together, the suppression of Let-7a by estradiol up-regulates integrin beta-3, vav3, and dicer. The increased Itgb3 expression promotes blastocyst attachment and intertwined with the up-regulated vav3 expression to enhance blastocyst outgrowth. The increased vav3 expression further stimulates cell proliferation and confers blastocyst invasion into the collagen matrix. Dicer, by altering microRNA processing, facilitates blastulation and thereby embryo implantation. Thus, the loss of Let-7a biological activities during blastulation is crucial to enhance blastulation and stimulate trophoblast activities for successful embryo implantation. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

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

Cervical cancer is the second major cancer among women worldwide and is associated with persistent infection of human papillomaviruses (HPVs). However, exposure to high-risk type HPVs alone is insufficient for tumor formation. Additional factors are required for the HPV-infected cervical cells to become tumorigenic. Activation of ß-catenin/TCF signaling is essential for transformation of HPV-immortalized keratinocyte into cancer. ß-catenin is excessively expressed in cervical cancer. Dysregulation of microRNAs is profoundly observed in various cancers but their roles in cervical cancer are obscure. MicroRNA-135a (miR-135a) regulates one of the negative regulators of ß-catenin signaling, E3 ubiquitin ligase Seven In Absentia Human Homolog 1 (SIAH1). A 39-fold increase in the expression of miR-135a occurs in early stage cervical cancer. This study hypothesized that over-expression of miR-135a transformed HPV-infected cervical cells to cancer by activating ß-catenin/TCF signaling through down-regulation of SIAH1. In this study, miR-135a was confirmed to be specifically up-regulated in cervical cancer tissues when compared with precancerous lesions. Force-expression of miR-135a induced tumorigenic properties (anchorage independent growth and metastatic abilities) in vitro of a non-tumorigenic cervical epithelial cell line NC104 immortalized by HPV-16 E6 and E7 oncoproteins (NC104 E6/E7). The metastatic activities induced by miR-135a required the presence of E6 and E7 proteins as the activities were not observed in another immortalized cervical cell-line from the same parental cells but without the oncoproteins. The observations were confirmed by the observations that miR-135a knockdown did not impair the above tumorigenic properties in a HPV-negative cervical cancer cell line, but suppressed them in HPV-positive cervical cancer cell lines. The mechanism of action of miR-135a in cervical cancer was evaluated. The tumorigenic effects was due to the inhibitory action of miR-135a on SIAH1 leading to up-regulation of ß-catenin/TCF signaling. MiR-135a force-expression enhanced the growth of the cervical cancer cell line HeLa and NC104 E6/E7-derived tumor in vivo. The effect of miR-135a was partially nullified by SIAH1 force-expression. These observations were in line with expression analyses in cervical biopsies, in which SIAH1 immunoreactivities were inversely correlated, whereas ß-catenin was positively correlated with the expression of miR-135a. The data illustrated an oncogenic role of miR-135a/SIAH1/ß-catenin signaling in cervical cancer formation. The role of miR-135a in the formation of cancer stem cells (CSCs) was also elucidated. The number of CD133+ cells was significantly higher in the miR-135a-transformed NC104 E6/E7 cells than the untreated group. The CD133+ cells isolated from the miR-135a-transformed NC104 E6/E7 possessed self-renewal, differentiation and multidrug resistance properties, up-regulation of miR-135a. They also expressed ß-catenin and the stemness genes OCT4, SSEA-4. CD133+ cells were also identified sporadically in fresh cervical tumors. The observations indicate that CD133+ cervical cancer cells possesses CSC properties. In conclusion, this thesis was the first to identify and characterize the functions of miR-135a as an oncomiR in cervical carcinogenesis. MiR-135a played a pivotal role in malignant transformation and cancer progression in HPV-infected cervical cells through miR-135a/SIAH1/ß-catenin signaling. The microRNA also enhanced the proportion of CD133-expressing cervical CSCs. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Cervical cancer is a common gynaecological malignancy worldwide, with more than 450,000 incidences every year. Its etiology has been well documented to be associated with persistent infection with high-risk genotypes of human papillomavirus (HPV). The carcinoma can be screened by convention Pap smear and liquid-based cytology. Although preventable, cervical cancer remains a primary cause of death from cancer in developing countries where cytological screening is not so available. In the past decades, many studies have been carried out to explore molecular screening or diagnosis of cervical cancer, such as HPV DNA testing, histological or cytological biomarkers. Micro RNAs, small non-coding RNA molecules of 18-25 nucleotides in length, areaberrantly expressed in various cancers. MiR-143 was reported consistently downregulated in cervical cancer tissues and cell lines, but its functional roles in cervical carcinogenesis has not been clearly illustrated. Ten miR-143 downstream target genes were chosen and their expression levels in five cervical cancer cell lines (HeLa, SiHa, CaSki, C4-I and C33A) were investigated. In general, the gene expressions of candidates are upregulated in our cell lines with lowmiR-143 level. To further identify specific miR-143 targets in cervical cancer for biomarkers, protein expressions of TARDBP, ERK5, KRAS and PHF6were significantly downregulated upon miR-143 overexpression. Hence, miR-143 level is inversely correlated with the mRNA and protein expressions of these target genes. Immunohistochemical study of ERK5 and TARDBP on FFPE samples including normal cervix, CINs and SCC cases showed that both ERK5 and TARDBP were positively stained in SCC samples, whereas weaker staining was found in CINs (both LSILs and HSILs) for both antigens. Thus, the intensity of positive staining ascended with the histological grading: LSIL, HSIL and SCC samples. Such differential expression pattern supports ERK5 and TARDBP as specific markers for high grade cancerous lesions. In summary, two targets of miR-143, ERK5 and TARDBP, could be specific markers for high-grade lesion of cervical cancer. This is supported by their transcript and protein expressions inversely associated with miR-143 level, and that their strong immunohistochemical positivity in SCC samples. Their underlying molecular mechanisms involved in carcinogenesis and possible future applications require more in-depth researches. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Hepatocellular carcinoma (HCC) is the major type of liver cancer. In Hong Kong, thousands of deaths are related to this disease every year. Hepatitis B virus (HBV) infection is one of the major risk factors of HCC development. The high prevalence of HBV carriers in Southeast Asia including Hong Kong can account for the particularly high HCC cases in these areas. HCC is often asymptomatic. The diagnosis and treatment are often delayed which lead to inapplicable of surgical resection. Meanwhile, conventional treatment regimes such as systemic chemotherapy were found to have limited responses. Hence, the case mortality rate of HCC is the second highest among all the cancers. Granulin-epithelin Precursor (GEP) is a glycoprotein growth factor which regulates multiple cellular functions. Our group has demonstrated that GEP is over-expressed in more than 70% of HCC cases and GEP expression is positively correlated to tumor malignancy. Our group has also verified that suppression of GEP by monoclonal antibody leads to significant inhibition of HCC growth and reduction of malignancy. Therefore, GEP has the potential to be a novel therapeutic target of HCC. MicroRNAs (miRNAs) are short non-coding RNAs that regulate mRNA translation. Previous studies showed that miRNA dys- regulation is closely associated with HCC progression and the high stability of miRNAs allows them to be cancer biomarkers or therapeutic targets. This project aims to investigate the miRNAs that regulate GEP and their functions in HCC. Potential GEP-regulating miRNAs were identified by literature review and in silico prediction by bioinformatics tools. MiR-615-5p, miR-588, miR-29b, miR-195, and miR-659 were identified as the potential candidates. Quantitative polymerase chain reaction (qPCR) was utilized to examine the miRNAs’ expressions in HCC clinical samples. Only miR-29b and miR-195 were detected and hence they were selected for further study. Our results showed that miR-29b and miR-195 expression levels were significantly decreased in HCC comparing to adjacent non‐tumor tissue (P<0.001) in more than 70% of cases. MiR‐195 and miR‐29b were over‐expressed in Hep3B HCC cell lines by miRNA mimics and GEP protein level was significantly suppressed after miR-29b mimic transfection. The transcript level of GEP was found to be unchanged after the miR‐29b over-expression. This suggests miR‐29b does not regulate GEP protein expression by mRNA degradation. The effects of miR‐195 and miR‐29b on HCC proliferation were also examined. The growths of HCC cells were suppressed notably after over-expression of miR‐195 (P<0.005) and miR‐29b (P<0.005) respectively. In conclusion, miR‐195 and miR‐29b are frequently down-regulated in HCC. MiR‐29b can negatively regulate GEP expression and does not interfere with GEP mRNA level. Furthermore, miR‐195 and miR-29b can function to inhibit HCC cell growth significantly. / published_or_final_version / Surgery / Master / Master of Philosophy

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The generation of synthetic oligonucleotides is dependent on an efficient solid-phase synthesis methodology. This thesis evaluates the 2'-O -levulinyl (Lv) and 2'-O-monomethoxytrityl (MMT) ribonucleosides, as possible synthons for RNA and branched RNA synthesis. A key feature of this RNA and bRNA synthesis procedure is their removal while still attached to the solid support and under conditions that prevent isomerization or cleavage of the nascent strands. For the first time, the stability of 3'-5'-internucleotide phosphate triesters (and diesters) adjacent to a ribose 2'-hydroxyl group was determined on a solid support. These studies are not only relevant to the proper assembly of branched and linear RNA species, but also to the stability of an unusual branched RNA species ("RNA X") proposed to form during the pre-mRNA splicing reactions in vitro. These studies are also important to the development of large quantities of native and chemically modified short interfering RNA (siRNA) for animal and human studies. / The 2'-O-Lv and 2'-O-MMT ribonucleoside monomers served as building blocks for the assembly of a series of branched nucleic acid species (bRNA, bDNA, msDNA and hyperbranched or "dendritic" DNA/RNA) with discrete length, base composition and structure. These structures were synthesized via an iterative divergent-growth strategy, which facilitates the regioselective branching, deblocking and chain lengthening steps from a branchpoint core. These structures served as useful materials (bio-probes) as demonstrated by the biological studies performed with E. coli RNaseH and the yeast lariat RNA debranching enzyme (yDBr1). These studies not only led to the identification of novel branched nucleic acid inhibitors of yDBR1 and RNase H, but also provided new insights about the substrate specificity of these important enzymes. / This thesis also describes the synthesis of a new nucleic acid form, the so-called "oxepane nucleic acids" (ONAs), in which the pentofuranose ring of DNA and RNA was replaced with a 7-membered heptose sugar ring. ONA were found to be much more resistant towards nuclease degradation than natural DNA, an important feature if these analogues are to be used in biological media. Furthermore, ONAs exhibited cross-pairing with complementary RNA and were found to elicit E. coli RNaseH mediated degradation of the RNA strand. These finding are significant because oligonucleotide-directed RNase H degradation of the target RNA is a key determinant for the gene-specific inhibitory potency of antisense oligonucleotides. When comparing the rates of RNase H-mediated degradation induced by 5 (furanose), 6 (2'-ene-pyranose) and 7 (oxepane) membered ring oligonucleotides, the following trend was observed: DNA > 2'-ene-pyranose NA > ONA. The implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme, particularly with regard to the required flexibility of the oligonucleotide strands that bind to the RNA target. Hence, ONAs are useful tools for biological studies and provide new insights into the structure/function of natural and alternative genetic systems.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 114-132) Also available in print.