By performing microRNA microarrays to globally detect any novel miRNAs in the limbus, eleven microRNAs (hsa-miR-136, hsa-miR-373*, hsa-miR-150, hsa-miR-143, hsa-miR-455, hsa-miR-145, hsa-miR-381, hsa-miR-224, hsa-miR-338, hsa-miR-154, hsa-miR-377) were found to be upregulated while two microRNAs (hsa-miR-122a and hsa-miR-425-3p) were identified as downregulated by more than 2 folds. Among these, hsa-miR-143 and hsa-miR-145 were distingushed to be the most significantly up-regulated limbal miRNAs. Individual assessement of the microarray results of a recently reported stem cell specific microRNA, hsa-miR-21, were also upregulated by more than two thousand fold when comparing limbus and cornea. miR-21, miR-143 and miR-145 were therefore selected as the most likely microRNA candidates in the present study. The expression level of these miRNA candidates were validated and confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To localize these candidates, we performed in situ hybridization on frozen corneal rim sections using locked nucleic acid (LNA)-modified oligonucleotide probes. Results showed that miR-2I, 143 and 145 were confined in the limbal region with gradation of expression level along the basal-suprabasal line. / Functional roles of these microRNAs were then deciphered by overexpressing human corneal epithelial cell line (HCE) with precursor microRNAs (pre-miRs) through lipophilic transfection. Results showed that high endogenous level of miR-145 could inhibit cell proliferation by 3.5 fold as shown from MTT proliferation assay at day 5, and could generate discrete spherical colonies that resembles the morphology of holoclones at day 8, but not the other two candidate miRNAs. / In conclusion, 1 have identified three novel microRNAs (hsa-miR-21, 143, 145) which were precisely upregulated in the limbus region, while miR-145 was being the most limbal specific. In addition, the functions of miR-145 were found to be inhibitory on cell proliferation, possibly through the indirect regulation of IFNB1. These unprecedented results may suggest a therapeutic potential of miR-145 on limbal stem cell deficiency and limbal tumors because miR-145 can affect cell survival and proliferation. / MicroRNAs is a family of small non-coding RNAs that, in human, binds imperfectly to the 3' untranslated region (UTR) of target mRNAs for translational repression or negative regulation. Recent studies have shown that such negative regulatory pathways may play pivotal roles in the maintenance of asymmetric cell division in embryonic and tissue specific stem cells. Human corneal epithelial progenitor cells (CEPC), a tissue specific stem cell lineage residing between cornea and conjunctiva in the Palisade of Vogt of the limbus region, is known to maintain corneal homeostasis throughout human life. They respond to injury and normal wearing by rapid proliferation and differentiation into transit amplifying cells (TACs) and eventually corneal epithelial cells, though the biological factors controlling this homeostatic switch are still largely unknown. Here I hypothesized that microRNAs can participate in CEPC regulation. Experiments elucidating the anatomical distribution and functional roles of microRNAs on the human cornea rims were performed to testify this proposition. / Protocols aim at enriching the CEPC population were then devised. For the first time a four parameter cell sorting system utilizing ABCG2, Connexin 43, Notch 1 and pyronin Y as markers was established for the prospective in vitro study. Nevertheless, manual microdissection isolating the limbus region and the cornea region was employed for the present study of microRNAs. / This study begins with the phenotypic validation of human cornea rims recruited from the Chinese Hong Kong population using immunohistochemistry. Conventional CEPC markers (p63, EGFR, cytochrome oxidase and cytokeratin 15), embryonic stem cell marker (stat1) and cancer stem cell markers (p73, MDM2 and pStat1) were expressed in the limbus region, suggesting that these specimens contained a source of CEPC for attesting our hypothesis. / To determine the mRNA targets of candidate microRNAs in HCE cells, Whole Human Genome Oligo Microarray Kits (Agilent Technologies) which contained 41K human genes and transcripts were employed. When compared to the scrambled control, HCE cells over-expressed with hsa-miR-21, 143 or 145 revealed differential expression of genes that participate in cell activation, motility and proliferation. Of note, interferon beta 1 fibroblast (IFNB1), a gene that is often deleted or rearranged in cancers, was significantly upregulated by a medium of 1093 fold in pre-miR-145 treated cells as confirmed by real time PCR assays. / Lee, Sharon Ka-wai. / "December 2009." / Advisers: Calvin Chi-Pui Pang; Gary Hin Fai Yam. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 216-252). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344453 |
| Date | January 2010 |
| Contributors | Lee, Ka Wai Sharon., Chinese University of Hong Kong Graduate School. Division of Ophthalmology and Visual Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xxv, 252 leaves : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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