Iroquois genes encode a family of highly conserved TALE homeodomain transcription factors that are involved in multiple developmental processes. Physiological tests indicated that Irx3 and Irx5 mutant mice displayed hearing impairment. However, the functions of these two genes during inner ear development are not known. The aim of this study is to characterize the roles of Irx3 and Irx5 during mammalian inner ear development using mouse models, in order to reveal the underlying mechanism for the hearing abnormality in the mutants. Two mouse mutants, Irx3tauLacZ and Irx3flox5EGFP with β-gal and EGFP reporters, were analyzed to examine the expression of these two genes in the otic vesicle and cochlear epithelium. In the otocyst, both Irx3 and Irx5 were expressed in the ventral-medial region. Irx5 expression was restricted to the non-sensory domain of the cochlear epithelia, while Irx3 was widely expressed, including the auditory sensory organ, the organ of Corti. The overlapping expression patterns of Irx3 and Irx5 suggest that they may share redundant functions. To investigate the roles of Irx3 and Irx5 during inner ear development, phenotypic analysis was performed on Irx3-/-, Irx5-/- and Irx3/5-/- mutant embryos. As shown by paint-filling analysis, Irx3/5-/- displayed shortened cochlear duct, enlarged cochlear lumen with fused sensory organ. Whole-mount phalloidin staining of hair cell bundles showed that Irx3-/- displayed occasional ectopic inner hair cells. Moreover, only supernumerary vestibular hair cell-like cells were developed in Irx3/5-/- mutant. These results suggest that Irx3 and Irx5 are required for inner ear morphogenesis and the formation of organ of Corti. To understand the effect of Irx3 and Irx5 in the cellular patterning of the cochlea, mutant cochleae were analyzed with markers for different regions of the cochlear epithelia. Altered expression domain of MyoVIIa, Sox2 and Gata2 in Irx3/5-/- cochlea revealed that the boundary between the Kolliker’s organ and the organ of Corti was lost and the location of sensory and non-sensory region was shifted. These results imply that Irx3 and Irx5 function in the establishment of the sensory/non-sensory boundary. It is known that p27kip1 regulates the wave of cell cycle exit in the developing organ of Corti and Sox2 takes part in prosensory specification. To explore the underlying reason for the patterning defects in Irx3/5-/- mutant, cochlear duct from prosensory stages were analyzed. Irx3/5-/- showed altered Sox2 and p27kip1 expression, with expanded prosensory domain and disrupted cell cycle exit. Ectopic prosensory proliferation was detected in the middle turn of the cochlear duct at E13.5 by BrdU incorporation assay. Therefore, Irx3 and Irx5 may participate in the subdivision of sensory territory in developing cochlea by controlling prosensory proliferation. In summary, this study demonstrates that Irx3 and Irx5 cooperate in multiple aspects of inner ear development: an early role to regulate prosensory proliferation and cell cycle exit; a second role to regulate cellular patterning of the cochlear duct by controlling the setting of sensory/non-sensory boundaries in the cochlea; a later role to regulate inner ear morphogenesis. This study supports the idea that Irx3 and Irx5 act as patterning genes during vertebrate evolution. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Epithelial ovarian cancer is a common female malignancy with a relatively high mortality rate worldwide. This may due to a lack of efficient diagnostic methods at early stage and worsen by complications caused by metastasis at advanced stage. For successful metastasis, cancer cells detached from the original growing sites have to survive in the body circulation before conquering a distant location within the body. Resistance to anoikis (apoptosis induced without appropriate extracellular matrix) is therefore utmost important during metastatic spreading. In addition, a pre-metastatic niche remodeled by cancer cells is also a pre-requisite for metastatic colonization. Emerging evidence has suggested the dysregulation of miRNAs is associated with different aspects of tumorigenesis. However, the specific roles of miRNAs in anoikis resistance and in remodeling of distant niche remain unknown thus far. This study attempted to investigate the functional roles of miR-141, in particularly anoikis resistance of ovarian cancer cells and the reprogramming of stromal cells. The miR-200 family is frequently upregulated and associated with human cancer metastasis. In this study, by cDNA array profiling together with biochemical and functional studies, miR-141, a member of miR-200 family, was identified as an oncomiR enhancing cell viability in low serum medium and anoikis resistance. Moreover, enforced expression of miR-141 led to bigger tumor sizes and promoted metastatic colonization in mouse models. Further studies demonstrated miR-141 directly targets tumor suppressive KLF12 in ovarian cancer cells, depletion of KLF12 could mimic function of miR-141. Clinical study revealed the upregulated miR-141 was significantly correlated with the downregulated KLF12, serous subtype, advanced and distant metastatic ovarian cancer. Furthermore, Genechip profiling, Human Apoptosis Array and Luciferase reporter assay revealed the upregulated miR-141 and downregulated KLF12 enhanced anoikis resistance via elevation of survivin which protect cells against intrinsic apoptotic activity. On the other aspect, miR-141 was found to be a secretary miRNA and commonly detected in the serum of ovarian cancer patients. The upregulated miR-141 expression was also correlated to levels of common cancer biomarker CA125. Importantly, the serum miR-141 level was significantly correlated with the tumor burden of patients during treatments, indicating it could be used as a non-invasive biomarker for ovarian cancers. Finally, based on miR-141 as tumor-secreted and circulated miRNA, a series of functional studies demonstrated that miR-141 could be transferred to hFF-1 fibroblast cells. Intriguingly, ovarian cancer cells cultured in miR-141-fibroblast culturing medium showed a remarkable increase of cell migration, suggesting that the remodeled-miR-141 fibroblast cells can secrete stimulating factors and promote ovarian cancer cells aggressiveness. This is the first study showing miR-141 could reprogram fibroblast cells to be a niche for ovarian cancer cell dissemination and metastatic progression. However, further investigations for verifying such functions are warranted. In conclusion, this study provides strong evidence that miR-141 is oncoMir enhancing ovarian cancer cell plasticity in metastasis e.g. anoikis resistance. Moreover, the finding of secretary form miR-141 not only gives the feasibility to be a potential biomarker for detecting ovarian cancer but also shows a possible mechanism of how miRNAs reprogram the distant niche for metastatic colonization. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Neuroblastoma is a childhood solid tumor of a unique propensity to either regress spontaneously or grow relentlessly. Emerging evidence indicated that neuroblastoma contains heterogeneous populations of cells, and commitment of these cells to neuronal lineage may result in aggressive progression in patients, whereas to fibromuscular lineage may give a favorable outcome. However, mechanism(s) controlling the lineage commitment of neuroblastoma cells remains to be identified. Our preliminary data suggested that Kr?ppel-Like Factor 4 (KLF4) might promote neuroblastoma regression. KLF4 is a transcription factor regulating a variety of cellular functions, including proliferation and cell cycle progression. Recent studies have demonstrated that KLF4 may act as both tumor suppressor and oncogene in a cell-context dependent manner. Importantly, our preliminary data showed that low KLF4 expression is highly associated with poor clinical outcomes of the neuroblastoma patients. In addition, we found that overexpression of KLF4 suppresses neuroblastoma cell growth accompanied with loss of tumorigenicity. Morphologically, KLF4 overexpressing cells changed their morphologies to become epithelial-like, strongly substrate-adherent and expressing smooth muscle marker. Therefore, we hypothesized that KLF4 exerts its effects through two ways, it may (i) function to inhibit cell growth and reduce tumorigenicity; and (ii) promote differentiation of the neuroblastoma cells to the non-tumorigenic, fibromuscular-like cells. RT-PCR data revealed the differential expression of KLF4 in 11 neuroblastoma cell lines. In particular, a modest expression was found in Be(2)C, a cell line which was formerly demonstrated to differentiate and form tumor in mice xenograft assay. It was therefore chosen as the study model. To assess the effects of KLF4 knockdown on tumor growth, stable knockdown clones from Be(2)C cells were established by lentiviral transduction of KLF4-targeting shRNA. In parallel, clones that stably expressed non-target shRNA were used as controls. After the transduction, two stable knockdown clones showing significant KLF4 downregulation were isolated from single colony (monoclonal stable clones) and a pool of cells (polyclonal stable clones) respectively. The cell proliferation and growth rate of the stable clones were then measured by 5-bromo-2’-deoxyuridine (BrdU) proliferation assay and growth curve assay. The results have indicated that both monoclonal and polyclonal stable KLF4 knockdown clones grow faster than the control clones. In order to examine the tumorigenicity in vivo, the stable clones were xenotransplanted to severe combined immunodeficient mice. The stable KLF4 knockdown clones showed a significant higher growth rate and formed a larger tumor. The stable clones were also treated with BrdU for four weeks for differentiation towards fibromuscular lineage. As anticipated, the control clones showed fibromuscular features, like more flattened and epithelial-like morphology. In contrast, the stable KLF4 knockdown clones failed to present the fibromuscular features after treatment. In addition, immunocytochemistry staining of SMA and quantitative analysis of the immunocytochemistry further confirmed that only the control clones showed higher SMA expression after BrdU treatment, while there is no change in the SMA expression in the stable KLF4 knockdown clones. These results demonstrated that KLF4 functioned by inhibiting neuroblastoma cell proliferation and growth, reducing the tumorigenicity, and it was required for fibromuscular differentiation. / published_or_final_version / Surgery / Master / Master of Philosophy

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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences