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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊曜宇(Eric Y. Chuang) | |
dc.contributor.author | Hua-Cheng Lo | en |
dc.contributor.author | 羅華成 | zh_TW |
dc.date.accessioned | 2021-06-15T13:32:31Z | - |
dc.date.available | 2020-09-02 | |
dc.date.copyright | 2020-09-02 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-20 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51387 | - |
dc.description.abstract | 良性前列腺增生(肥大)為前列腺最常見之良性疾患,但致病機轉的分子/細胞因子仍未清楚。慢性發炎除了可能是良性前列腺增生的重要因子外,對前列腺癌也可能扮演重要角色。本論文旨在運用基因體方法來研究良性前列腺增生與前列腺癌兩種前列腺疾病之新療法。本論文主分為兩部分。第一部分是找出慢性發炎與前列腺增生進展機轉相關的生物標記。首先收集並分析病患檢體及其臨床資訊,結果發現前列腺增生檢體發炎情形普遍,且與血清PSA值和前列腺增生體積有正相關。進一步將病人分成兩組:第一組,前列腺體積≦ 40 ml,第二組,前列腺體積> 40 ml,並利用微陣列分析發現:免疫和發炎相關路徑的基因表現在第二組有顯著向上調控。同時發現基因IL27RA在第二組呈現向下調控。此結果經免疫組織化學染色驗證。故我們假設IL-27RA與慢性發炎的調控與前列腺增生進展有關,並設計細胞生物實驗來驗證。結果顯示在BPH-1與PC-3細胞上,IL-27/IL-27RA訊息可抑制LPS/TLR4途徑活化後誘發之具促發炎與促增生的細胞激素(IL-6和IL-8)表現。總結IL-27/IL-27RA訊息可扮演調控慢性發炎與前列腺增生進展的角色。 論文第二部分則是著重在前列腺癌的研究。前列腺癌是排名第三常見造成男性死亡的惡性腫瘤,其治療涵蓋藥物,手術及放射治療。放射治療不僅能治癒早期前列腺癌,在晚期或末期骨轉移病患,也能達到控制疾病或緩解症狀的效果。然而部分前列腺癌病患有出現放射抗性(radioresistance),造成腫瘤復發與治療挑戰。前列腺癌放射抗性機轉未明。研究顯示微小核糖核酸(microRNA)可經由調控放射線引發之去氧核糖核酸傷害反應(DNA damage response)而改變放射敏感性(radiosensitivity)。以文獻回顧和網路資料庫分析發現microRNA-107(miR-107)在前列腺癌細胞受到放射線照射時的表現量下降,而顆粒體(granulin,GRN)為miR-107之標的基因。因此我們使用miR-107,miR-107抑制劑,shGRN與GRN過度活化等方式,發現miR-107能藉抑制GRN來增進PC-3放射敏感度。透過流式細胞儀和西方墨點法,我們發現活化miR-107能抑制p21和磷酸化CHK2的表現量,導致細胞週期G1/S期的休止與G2/M期的通過,最終造成PC-3的延遲性凋亡(delayed apoptosis)。 本論文涵蓋應用生物晶片與網路資料庫分析,找出良性前列腺增生重要生物標記並加以驗證;與探討微小核糖核酸調控前列腺癌放射抗性之機轉獲得新發現。藉由解譯前列腺疾病的基因體資訊與基因的變異性,了解相關分子表現型與路徑的改變,將有助於研究、發展前列腺疾病新療法。 | zh_TW |
dc.description.abstract | This thesis aims to use genomic approaches to investigate novel therapies for two prostate diseases, benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The thesis is presented in two parts. BPH is the most common benign prostatic disease in elder men. The underlying molecular/cellular factors of pathogenesis are still unclear. Chronic inflammation (CI) could play important roles. To identify biomarkers in the mechanism of CI leading to BPH progression, we analyzed correlations between BPH patients’ tissues and clinical information. The thirty BPH patients were divided into two groups: Group 1, volume ≦40 ml, and Group 2, volume >40 ml. Microarray analysis showed immune/inflammation-associated genetic pathways were significantly upregulated in the Group 2. Moreover, the gene IL27RA was downregulated in Group 2 and validated by immunohistochemical stain from tissue array. We further studied potential mechanisms of IL-27RA in chronic inflammation and BPH progression and verified that by in vitro cell models. The proliferative and proinflammatory cytokines (IL-6, IL-8) induced by LPS/TLR4 pathways were suppressed by IL-27/IL-27RA signaling in BPH-1 and PC-3 cells. We concluded IL-27/IL-27RA signaling may modulate chronic inflammation and BPH progression. The second part mainly focus on how to improve PCa treatment. PCa is the third leading cause of cancer deaths in men. Radiotherapy can cure most of PCa patients at early stages, and offer palliative effects at advanced/end stages. However, radioresistance leads to tumor recurrence and challenges. Studies showed microRNAs could modulate radiosensitivity by regulating radiation-induced DNA damages. By literature review and web databases survey, we found that miR-107 is downregulated in PCa cells after irradiation, and granulin (GRN) is a target gene of miR-107. The PC-3 cells were used to validate that miR-107 was downregulated, and GRN was upregulated in the cells with radiation exposure. By using miR-107 mimic, miR-107 inhibitor, shGRN and GRN overexpression, we concluded that overexpressed miR-107 may enhance radiosensitivity in PC-3 cells by suppressing GRN. Furthermore, we found that miR-107 may inhibit p21 and phosphorylate CHK2, lead to G1/S arrest and G2/M transit, and result in delayed apoptosis. We applied these genomic approaches to identify genetic variants of BPH and unveiled a novel mechanism in regulating PCa radiosensitivity. Decrypting the genomic data and genetic variants of molecular phenotypes and altered pathways will aid greatly to investigate novel therapies in prostate diseases. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T13:32:31Z (GMT). No. of bitstreams: 1 U0001-1008202009451800.pdf: 5371040 bytes, checksum: eabf0f4be7aa2c2f14ac43c97ce42de0 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 誌謝………………………………………………………………… i 中文摘要…………………………………………………………… ii Abstract…………………………………………………………… iv List of Figures…………………………………………………… xi List of Tables…………………………………………………… xv Chapter 1: Introduction……………………………… 1 1.1 The Prostate…………………………………………………… 1 1.2 Zones of the Prostate……………………………………… 1 1.3 Histophysiology of the Prostate………………………… 3 1.4 Diseases of the Prostate Gland…………………………… 5 1.4.1 Benign Prostatic Hyperplasia (BPH)……………… 5 1.4.2 Prostate Cancer (PCa) ……………………………… 8 1.5 Genomic Approaches…………………………………………… 11 1.5.1 Microarray Analysis…………………………………… 11 1.5.2 MicroRNA………………………………………………… 14 1.6 Overview of Objectives……………………………………… 16 Chapter 2: IL-27/IL-27RA Signaling May Modulate Inflammation and Progression of BPH via Suppressing the LPS/TLR4 Pathway……… 18 2.1 Introduction………………………………………………… 18 2.2 Materials and Methods……………………………………… 24 2.2.1 Collection of patient data and samples, ethics statement, and grouping… 24 2.2.2 Isolation and labeling of total RNA for gene expression profiling………………… 25 2.2.3 Data mining and statistical analysis…………………… 25 2.2.4 Quantitative reverse transcriptase-PCR………………… 26 2.2.5 Immunohistochemical staining………………………… 27 2.2.6 Chemicals and antibodies……………………………… 27 2.2.7 Cell culture and treatment……………………………… 28 2.2.8 Western blotting……………………………………… 29 2.2.9 Enzyme-linked immunosorbent assay (ELISA) analysis. 30 2.2.10 Statistical Analysis…………………………………… 30 2.3 Results……………………………………………………… 32 2.3.1 Clinical characteristics of BPH patients……………… 32 2.3.2 Gene expression profiling in BPH tissues…………… 34 2.3.3 Differential expression of immune-inflammation signaling Pathways between two groups of BPH patients……… 34 2.3.4 Down-regulation of IL27RA in BPH tissues in Group 2. 37 2.3.5 Constitutive Expression of IL-27RA and TLR4 in human prostate cells………… 43 2.3.6 LPS induced IL-6 and IL-8 production in human prostate epithelial cells, BPH-1 and PC-3……………… 46 2.3.7 Suppression of LPS-induced IL-6 and IL-8 expression by IL-27/IL-27RA signaling………………………… 47 2.3.8 Activation of IL-27/IL-27RA signaling can lead to phosphorylation of STAT1 and STAT3………………… 51 2.4 Discussion and Conclusion………………………………… 57 Chapter 3: MicroRNA-107 Enhances Radiosensitivity by Suppressing Granulin in PC-3 Prostate Cancer Cells……………… 66 3.1 Introduction………………………………………………… 66 3.2 Materials and Methods……………………………………… 71 3.2.1 Cell culture and irradiation treatment………………… 71 3.2.2 Cell infection and transfection………………………… 71 3.2.3 Virus production and cell infection…………………… 72 3.2.4 Clonogenic assay……………………………………… 73 3.2.5 Western blotting……………………………………… 74 3.2.6 RNA extraction, reverse transcription, and qRT-PCR quantification………………… 74 3.2.7 Cell proliferation assay………………………………… 76 3.2.8 Flow cytometry analysis of the cell cycle……………… 76 3.2.9 Annexin V analysis for apoptosis……………………… 77 3.2.10 Statistical analysis…………………………………… 77 3.3 Results……………………………………………………… 78 3.3.1 Altered expression of miR-107 in response to radiation in PC-3 cells……… 78 3.3.2 Overexpression of miR-107 inhibited proliferation and Enhanced radiosensitivity of PC-3 cells……………… 78 3.3.3 Granulin is a direct target of miR-107 and suppressed by miR-107 in PC-3 cells………………………………… 79 3.3.4 GRN expression increases in response to irradiation in PC-3 cells……………… 91 3.3.5 Knockdown of GRN sensitizes PC-3 cells to IR……… 91 3.3.6 GRN overexpression rescues proliferation and attenuates radiosensitivity induced by miR-107 in PC-3 cells… 95 3.3.7 MiR-107 alters cell cycle distribution and enhances delayed apoptosis in PC-3 cells……………………………… 102 3.4 Discussion and Conclusion………………………………… 110 Chapter 4: Conclusions and Future Perspectives………… 117 4.1 Conclusion…………………………………………………… 117 4.2 Future Perspectives………………………………………… 119 References…………………………………………………………… 122 Appendices…………………………………………………………… 133 | |
dc.language.iso | en | |
dc.title | 以基因體方法研究前列腺疾病新療法 | zh_TW |
dc.title | Genomic Approaches to Investigate Novel Therapies in Prostate Diseases | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 博士 | |
dc.contributor.author-orcid | 0000-0002-8396-8038 | |
dc.contributor.oralexamcommittee | 歐陽彥正(Yen-Jen Oyang),蔡孟勳(Mong-Hsun Tsai),李心予(Hsin-Yu Lee),賴亮全(Liang-Chuan Lai),查岱龍(Tai-Lung Cha) | |
dc.subject.keyword | 良性前列腺增生,慢性發炎,微陣列分析,前列腺癌,放射敏感性,微小核糖核酸, | zh_TW |
dc.subject.keyword | benign prostatic hyperplasia,chronic inflammation,microarray analysis,prostate cancer,radiosensitivity,microRNA, | en |
dc.relation.page | 133 | |
dc.identifier.doi | 10.6342/NTU202002763 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-08-20 | |
dc.contributor.author-college | 電機資訊學院 | zh_TW |
dc.contributor.author-dept | 生醫電子與資訊學研究所 | zh_TW |
顯示於系所單位: | 生醫電子與資訊學研究所 |
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