MicroRNA（miRNAs）是一類細小的非編碼RNA（ncRNA），能透過轉錄後機制調節靶標基因的表達。miRNA的發現，不僅提出一個嶄新的基因調節機制，更強調了小ncRNA於不同的生理和發展過程中的重要性。最近的研究更進一步展示了miRNA失調與癌症發展之間的因果關係。 / 我們此前曾利用陣列分析，發現miRNA在肝細胞癌（HCC）中的失調模式，揭示了miR-145在HCC的普遍下調。在本論文的第一部分，定量逆轉錄聚合酶鏈反應（qRT-PCR）進一步證實了miR-145在50的肝細胞癌患者（n=80）的腫瘤中出現表達下調，而且miR-145的表達下調更與較短的无病生存期相關。其中一個低內源性miR-145的肝癌腫瘤樣本被建立為細胞株─HKCI-C2。此體外模型保持低miR-145水平，並於恢復miR-145表達後，抑製細胞存活和增殖。多個計算機演算法均預測了miR-145可針對胰島素樣生長因子（IGF）信號通路中的多個基因，包括胰島素受體底物（IRS1）-1，IRS2和胰島素樣生長因子1受體。這些假定目標的蛋白表達亦被miR-145下調。熒光素酶檢測進一步驗證了miR-145和IRS1/IRS2 3'-非編碼區的直接目標關聯。隨後的分析也確定miR-145能下調 IGF信號通路下游的信號傳導，即活性β-catenin水平。 / 最近出現的深度測序技術，為研究miRNome提供了一個前所未有的平台，以識別已知和新的miRNA。此外，現代生物信息學技術可同時對不同類型的小ncRNA，如PIWI-interacting RNA（piRNAs）進行分析。在本論文的第二部分中，我們利用Illumina大規模並行測序對兩個肝癌細胞株（HKCI-4和HKCI-8）和正常肝細胞株（MIHA）的小RNA轉錄組進行研究。生物信息學和生物功能分析揭示一種新型piRNA（取名為piR-Hep1）在肝腫瘤發生中的重要角色。在73例肝癌中，qRT-PCR結果顯示piR-Hep1在47的肝癌組織出現上調。PiR-Hep1的沉默能抑制肝癌細胞存活、遷移和侵襲，同時亦減少了Akt的磷酸化。在miRNA的分析中，miR-1323被發現在肝癌組織中大量表達，並與肝硬化背景下產生的肝腫瘤相關。此外，miR-1323出現過表達的肝硬化肝癌患者的無病和整體存活率亦較差（P<0.009）。 / 總觀來說，本論文首次發現miR-145可同時抑制引致肝癌的IGF信號通路中的多個傳導因子，亦突出了piR-Hep1的功能重要性和miR-1323在肝癌患者中的預後意義。此外，本研究表明，傳統的陣列分析和新一代的測序技術均能發現重要的miRNA。新一代測序技術對轉錄組的全面分析，將對研究各種不同類型的ncRNAs在肝癌發生發展過程中的參與提供新的思路。 / MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNA) that post-transcriptionally regulate gene expression. The discovery of miRNAs not only puts forth an alternate gene regulatory mechanism, but also underscores the importance of small ncRNAs as pivotal regulators of diverse physiological and developmental processes. Recent studies have emphasized a causal link between miRNA deregulation and cancer development. / Our group has previously reported on dysregulated miRNA pattern in hepatocellular carcinoma (HCC) by array-based profiling, which revealed common downregulation of miR-145. In the first part of this thesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) corroborated reduced miR-145 expression in 50% of tumors in a cohort of 80 HCC patients, which also correlated reduced miR-145 expression with shorter disease-free survival of patients. One HCC tumor analyzed with low endogenous miR-145 was propagated as cell line. This in vitro model HKCI-C2 maintained low miR-145 level and upon restoration of miR-145 expression, a consistent inhibitory effect on cell viability and proliferation was readily observed. Multiple in silico algorithms predicted that miR-145 could target a number of genes along the insulin-like growth factor (IGF) signaling, including insulin receptor substrate (IRS1)-1, IRS2 and insulin-like growth factor 1 receptor. Protein expression of these putative targets was concordantly downregulated in the presence of miR-145. Luciferase reporter assay further verified direct target association of miR-145 to specific sites of IRS1 and IRS2 3’-untranslated regions. Subsequent analysis also affirmed the modulation of IGF signaling cascade by miR-145 as evident by reduction of the downstream mediator, namely, the active β-catenin level. / The recent advent of deep sequencing has provided an unprecedented platform to study the miRNome, in which both known and novel miRNAs can be identified. Moreover, bioinformatics advances have enabled different types of small ncRNAs, e.g. piwi-interacting RNAs (piRNAs), to be analyzed simultaneously. In the second part of this thesis, small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Combined bioinformatic and biological analyses revealed the involvement of a novel piRNA, designated as piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be up-regulated in 47% of HCC in a cohort of 73 HCC patients by qRT-PCR. Silencing of piR-Hep1 inhibited cell viability, motility and invasiveness with a concomitant reduction of Akt phosphorylation. In the analysis of miRNA, miR-1323 was found to be abundantly expressed in HCC and distinctly associated with tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic-HCC correlated with poorer disease-free and overall survivals of patients (P<0.009). / Taken together, results from this thesis showed for the first time the pleiotropic effect of miR-145 on targeting multiple components of the oncogenic IGF signaling pathway in HCC. In addition, the functional importance of piR-Hep1 and the prognostic significance of miR-1323 in HCC were highlighted. Studies conducted demonstrated that important miRNAs can be discovered by both traditional array-based profiling and next-generation sequencing. Moreover, comprehensive definition of transcriptome by next-generation sequencing unveils virtually all types of ncRNAs and provides new insight into the liver carcinogenic events. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Law, Tak Yin. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 180-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts also in Chinese. / Acknowledgements --- p.i / Publications --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Contents --- p.viii / List of Figures --- p.xiii / List of Tables --- p.xv / Abbreviations --- p.xvi / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma - One of the world’s most deadly killers --- p.2 / Chapter 1.2 --- MicroRNAs - a tiny molecule with enormous impacts --- p.10 / Chapter 1.2.1 --- Discovery of miRNAs --- p.11 / Chapter 1.2.2 --- Biogenesis and actions of miRNA --- p.13 / Chapter 1.3 --- MiRNAs and cancer --- p.16 / Chapter 1.4 --- Involvements of miRNAs in HCC etiological factors --- p.18 / Chapter 1.4.1 --- Viral hepatitis infection --- p.19 / Chapter 1.4.2 --- Chronic heavy alcohol consumption --- p.26 / Chapter 1.4.3 --- Dietary aflatoxin exposure --- p.28 / Chapter 1.4.4 --- Male gender --- p.31 / Chapter 1.4.5 --- Obesity --- p.33 / Chapter 1.5 --- Regulation of cancer-associated signaling network by microRNAs --- p.34 / Chapter 1.5.1 --- Apoptotic pathway --- p.37 / Chapter 1.5.1.1 --- Intrinsic pathway --- p.38 / Chapter 1.5.1.2 --- Extrinsic pathway --- p.39 / Chapter 1.5.2 --- Cell cycle regulators --- p.41 / Chapter 1.5.2.1 --- G₁/S transition --- p.42 / Chapter 1.5.2.2 --- G₂/M transition --- p.43 / Chapter 1.5.3 --- Receptor tyrosine kinase-mediated pathways --- p.45 / Chapter 1.5.3.1 --- c-MET-activated signaling --- p.45 / Chapter 1.5.3.2 --- PI3K-Akt --- p.47 / Chapter 1.5.3.3 --- RAS-RAF-MEK-ERK cascade --- p.48 / Chapter 1.5.4 --- TGF-ß signaling pathways --- p.50 / Chapter 1.5.5 --- Metastatic pathways --- p.52 / Chapter 1.5.5.1 --- MiRNAs with metastatic suppressing effects --- p.52 / Chapter 1.5.5.2 --- MiRNAs with metastatic promoting effects --- p.53 / Chapter 1.6 --- Clinical potentials of microRNAs - a killer or a cure? --- p.56 / Chapter 1.6.1 --- MiRNAs involvements in HCC risk prediction --- p.57 / Chapter 1.6.2 --- MiRNAs as diagnostic biomarkers --- p.59 / Chapter 1.6.3 --- MiRNAs as prognostic biomarkers --- p.60 / Chapter 1.6.4 --- Effects of miRNAs on responses to therapy --- p.61 / Chapter 1.7 --- Non-coding RNAs --- p.62 / Chapter 1.8 --- Aims of study --- p.63 / Chapter 2 --- Materials and Methods --- p.65 / Chapter 2.1 --- Quantitative reverse transcription polymerase chain reaction (qRT-PCR) --- p.66 / Chapter 2.1.1 --- Materials --- p.66 / Chapter 2.1.1.1 --- Total RNA extraction --- p.66 / Chapter 2.1.1.2 --- DNase treatment --- p.66 / Chapter 2.1.1.3 --- Reverse transcription --- p.66 / Chapter 2.1.1.4 --- Quantitative polymerase chain reaction --- p.66 / Chapter 2.1.2 --- Methods --- p.67 / Chapter 2.1.2.1 --- Total RNA extraction --- p.67 / Chapter 2.1.2.2 --- DNase treatment --- p.68 / Chapter 2.1.2.3 --- Reverse transcription --- p.69 / Chapter 2.1.2.4 --- Quantitative polymerase chain reaction --- p.69 / Chapter 2.2 --- Transfection --- p.70 / Chapter 2.2.1 --- Materials --- p.70 / Chapter 2.2.2 --- Methods --- p.70 / Chapter 2.2.2.1 --- Evaluation of HCC cells transfection efficiency --- p.70 / Chapter 2.2.2.2 --- Transfection --- p.71 / Chapter 2.3 --- In vitro functional assay --- p.72 / Chapter 2.3.1 --- Materials --- p.72 / Chapter 2.3.1.1 --- Cell viability assay --- p.72 / Chapter 2.3.1.2 --- Colony formation assay --- p.72 / Chapter 2.3.1.3 --- Cell cycle analysis --- p.72 / Chapter 2.3.1.4 --- Apoptosis assay --- p.72 / Chapter 2.3.1.5 --- Cell motility and invasion assay --- p.73 / Chapter 2.3.2 --- Methods --- p.73 / Chapter 2.3.2.1 --- Cell viability assay --- p.73 / Chapter 2.3.2.2 --- Colony formation assay --- p.74 / Chapter 2.3.2.3 --- Cell cycle analysis --- p.75 / Chapter 2.3.2.4 --- Apoptosis assay --- p.75 / Chapter 2.3.2.5 --- Cell motility and invasion assay --- p.76 / Chapter 2.4 --- Luciferase reporter assay --- p.78 / Chapter 2.4.1 --- Materials --- p.78 / Chapter 2.4.1.1 --- Cloning --- p.78 / Chapter 2.4.1.2 --- Cycle sequencing --- p.78 / Chapter 2.4.1.3 --- Luciferase reporter assay --- p.79 / Chapter 2.4.2 --- Methods --- p.79 / Chapter 2.4.1.1 --- Cloning --- p.79 / Chapter 2.4.2.2 --- Cycle sequencing --- p.81 / Chapter 2.4.2.3 --- Luciferase reporter assay --- p.82 / Chapter 2.5 --- Western blot --- p.84 / Chapter 2.5.1 --- Materials --- p.84 / Chapter 2.5.2 --- Methods --- p.85 / Chapter 2.5.2.1 --- Cell harvesting and protein quantitation --- p.86 / Chapter 2.5.2.2 --- Western blotting --- p.86 / Chapter 2.6 --- Small RNA Sequencing --- p.88 / Chapter 2.6.1 --- Materials --- p.88 / Chapter 2.6.2 --- Methods --- p.88 / Chapter 2.6.2.1 --- Sample preparation --- p.88 / Chapter 2.6.2.2 --- Cluster generation by bridge amplification --- p.88 / Chapter 2.6.2.3 --- Sequencing by synthesis --- p.89 / Chapter 2.7 --- Northern blot analysis --- p.94 / Chapter 2.7.1 --- Materials --- p.94 / Chapter 2.7.2 --- Methods --- p.94 / Chapter 2.7.2.1 --- Polyacrylamide gel electrophoresis (PAGE) --- p.94 / Chapter 2.7.2.2 --- Probe preparation --- p.95 / Chapter 2.7.2.3 --- Hybridization, stringency washes and signal detection --- p.95 / Chapter 3 --- Conventional miRNA profiling reveals miR-145 as a tumor suppressor in HCC --- p.97 / Chapter 3.1 --- Introduction --- p.98 / Chapter 3.2 --- Materials and Methods --- p.102 / Chapter 3.2.1 --- Patients --- p.102 / Chapter 3.2.2 --- qRT-PCR --- p.104 / Chapter 3.2.3 --- Cell line --- p.105 / Chapter 3.2.4 --- Transfection --- p.106 / Chapter 3.2.5 --- In vitro functional assay --- p.107 / Chapter 3.2.5.1 --- Cell viability assay --- p.107 / Chapter 3.2.5.2 --- Colony formation assay --- p.107 / Chapter 3.2.5.3 --- Flow cytometry assay --- p.107 / Chapter 3.2.6 --- miRNA target prediction --- p.109 / Chapter 3.2.7 --- Luciferase reporter assay --- p.110 / Chapter 3.2.8 --- Western blot --- p.112 / Chapter 3.2.9 --- Immunohistochemistry --- p.113 / Chapter 3.2.10 --- Statistical analysis --- p.114 / Chapter 3.3 --- Results --- p.115 / Chapter 3.3.1 --- Down-regulation of miR-145 in primary HCC --- p.115 / Chapter 3.3.2 --- Re-expression of miR-145 induced G₂-M arrest and apoptosis --- p.119 / Chapter 3.3.3 --- IRS1, IRS2 and IGF1R expressions --- p.124 / Chapter 3.3.4 --- miR-145 targeted both IRS1 and IRS2 and elicited IGF signaling --- p.126 / Chapter 3.4 --- Discussion --- p.131 / Chapter 4 --- Small RNA Deep sequencing reveals novel non-coding RNAs in HCC --- p.134 / Chapter 4.1 --- Introduction --- p.135 / Chapter 4.2 --- Materials and Methods --- p.136 / Chapter 4.2.1 --- Cell lines --- p.136 / Chapter 4.2.2 --- Patients --- p.137 / Chapter 4.2.3 --- Small RNA Sequencing --- p.139 / Chapter 4.2.4 --- Bioinformatics analysis --- p.140 / Chapter 4.2.4.1 --- Sequence mapping and ncRNA identification --- p.140 / Chapter 4.2.4.2 --- Putative miRNA prediction --- p.140 / Chapter 4.2.4.3 --- Putative piRNA identification --- p.140 / Chapter 4.2.4.4 --- Differentially-expressed ncRNAs identification --- p.141 / Chapter 4.2.5 --- qRT-PCR --- p.142 / Chapter 4.2.6 --- Northern blot analysis --- p.143 / Chapter 4.2.7 --- Transfection --- p.144 / Chapter 4.2.8 --- In vitro functional assays --- p.145 / Chapter 4.2.8.1 --- Cell viability assay --- p.145 / Chapter 4.2.8.2 --- Cell motility and invasion assay --- p.145 / Chapter 4.2.9 --- Western blot analysis --- p.147 / Chapter 4.2.10 --- Statistical analysis --- p.148 / Chapter 4.3 --- Results --- p.149 / Chapter 4.3.1 --- Small RNA Sequencing --- p.149 / Chapter 4.3.2 --- Up-regulation of putative piR-Hep1 in HCC --- p.155 / Chapter 4.3.3 --- piR-Hep1 silencing reduced cell viability and invasiveness --- p.159 / Chapter 4.3.4 --- Novel miR-1323 overexpression in HCC --- p.162 / Chapter 4.4 --- Discussion --- p.171 / Chapter 5 --- Concluding remarks and future perspectives --- p.175 / Chapter 6 --- References --- p.179
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328209 |
| Date | January 2013 |
| Contributors | Law, Tak Yin., Chinese University of Hong Kong Graduate School. Division of Anatomical and Cellular Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xviii, 200 leaves) : ill. (some col.), col. maps |
| 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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