泌尿道上皮癌相關的miRNAs在膀胱癌之研究

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姓名

方培倫(Pei-Luen Fang) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

泌尿道上皮癌相關的miRNAs在膀胱癌之研究
(Study of urothelial carcinoma-related miRNAs in bladder cancer)

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至系統瀏覽論文 (2024-8-1以後開放)

摘要(中)

在泌尿系統中，泌尿道上皮癌（Urothelial carcinoma, UC）是一種常見的惡性疾病，由病變的泌尿上皮所衍生而成，過去又稱作移行上皮細胞癌，通常伴隨著較差的預後，且有高達90%的膀胱癌（Bladder cancer, BC）病患都是泌尿道上皮癌患者。一般來說，泌尿道上皮癌發生在男性身上的機率較女性高，在年長者中機率較年輕人高。在台灣的病患中，泌尿道上皮癌也是最常見的泌尿道腫瘤，然而泌尿道上皮癌卻很難在早期被發現。經統計發現，慢性腎臟病人患有泌尿道上皮癌的機率是一般人的七倍；洗腎病人患有泌尿道上皮癌的機率是一般人的兩、三百倍。
在我們實驗室先前的研究結果指出，在慢性腎臟病的患者和同時患有慢性腎臟病、泌尿道上皮癌的患者中，以及洗腎病人和患有泌尿道上皮癌的洗腎病人中，有一些miRNA的表達有顯著差異，我們將這些miRNA作為接下來進行研究的候選miRNA共八個。
首先，從ArrayExpress數據庫中搜尋包含miRNA表現量和存活率且與膀胱癌相關的數據集，再透過預測網站預測miRNA可能的標靶基因。經分析後，我們發現只有一個候選miRNA的表現量高低對BC病患的存活有顯著影響，miR-19b-1-5p表現量較高的病患存活率較佳，可能扮演抑制腫瘤的腳色。此外，透過miRNA可能標靶基因的表現量高低分群，發現其中四個miRNA標靶基因的表現量高低對於病患存活率有顯著差異的影響，其中包括miR-19b-1-5p的標靶基因EDN1基因。
再者，我們為了調查候選miRNA對BC的影響，因此對兩株膀胱癌細胞株 (RT4, J82) 進行轉染，發現當候選miRNA過度表達時，J82和RT4細胞的生長速率沒有明顯下降，但在過度表達miR-19a-5p和miR-155-5p時，J82細胞群體數量顯著減少。另外，我們也觀察到當miR-150-5p過度表達時，會抑制J82細胞的遷移爬行能力；當miR-19a-5p、miR-30a-5p、miR-155-5p過度表達時，會抑制J82細胞的侵襲能力。這些結果說明候選miRNA可能會參與抑癌作用，但miR-19b-1-5p並不會抑制腫瘤的細胞增生、爬行與侵襲。
最後，為了探討survival和functional test結果不同的原因，我們透過GEO數據庫和RT-qPCR的方法調查候選miRNA在病人血清和細胞株中的表現，發現大部分的候選miRNA在BC有較高的表現，說明了UC相關的miRNA可能參與致癌作用。總而言之，這些miRNA在BC中扮演重要的腳色，但在不同的樣本中為何有不同的表現，這點會再進一步調查。

摘要(英)

Bladder cancer (BC) is a common malignant disease with poor prognosis in urinary system, which occurs in men more often than women and usually affects older adults. About 90% of bladder cancer is urothelial carcinoma (UC) which is known as transitional cell carcinoma (TCC) previously. UC is the most common urinary tract tumor in Taiwan patients. However, it is hard to be diagnosed at early stage. In addition, it has been found that the risk of developing UC in chronic kidney disease (CKD) patients is seven times higher than in normal people and the risk of developing UC in hemodialysis (HD) patients is 200-300 times higher than in normal people.
Our previous data showed that the expression of several miRNAs in urine and plasma were significant different between CKD patients and CKD patients with UC, or between HD patients and HD patients with UC. We chose these miRNAs as our candidate miRNAs to study their roles in BC.
Firstly, we analyzed the data of candidate miRNAs from ArrayExpress database and predicted the targets from four websites. Then, we obtained the survival rate curves of patients. The results showed that only the expression of miR-19b-1-5p significantly regulate survival of BC patients. Patients with high expression of miR-19b-1-5p had significantly longer overall survival than those with low expression. miR-19b-1-5p might act as a tumor suppressor. The expression of four miRNAs target genes significantly regulate survival of BC patients. Most interestingly, miR-19b-1-5p appeared to target EDN1 gene to have an impact on overall survival in BC patients.
Furthermore, we transfected negative control (NC) or candidate miRNA mimics in two BC cells (J82 and RT4) in order to know the function of these miRNAs in BC. We found that overexpression of candidate miRNAs could not significantly inhibit cell proliferation in J82 and RT4 cells. But, overexpression of miR-19a-5p and miR-155-5p could significantly decrease cell colonies in J82 cells. We also observed that overexpression of miR-150-5p could inhibit migration in J82 cells and overexpression of miR-19a-5p, miR-30a-5p, and miR-155-5p could inhibit invasion in J82 cells. The data indicated that these miRNAs might be associated with BC anti-carcinogenesis, but overexpression of miR-19b-1-5p did not inhibit cell proliferation, colony formation, migration and invasion.
Finally, because of different results from survival curves and functional tests, we investigated the expression of candidate miRNAs in patients’ serum and cells through GEO database and RT-qPCR analysis. We found that almost of candidate miRNAs have higher expression in BC. The data might indicate that UC-related miRNAs were involved in BC carcinogenesis, and we will further investigate the reasons for different results.

關鍵字(中)

★ 泌尿道上皮癌
★ 膀胱癌

關鍵字(英)

★ miRNA
★ urothelial carcinoma
★ bladder cancer

論文目次

摘要 v
Abstract vii
致謝 ix
目錄 xi
List of figures and table xiii
Abbreviation list xiv
I. Introduction 1
1. Urothelial carcinoma (UC) 1
1.1 Epidemiology 1
1.2 Etiology 2
1.3 Treatment 2
2. miRNA 2
2.1 Biogenesis and mechanism of miRNAs 2
2.2 Mechanisms of miRNA regulation 3
2.3 miRNA dysregulation in cancer 4
2.4 miRNA application in cancer 5
3. Purpose and significance 6
3.1 To investigate the functions of candidate miRNAs in bladder cancer 6
II. Materials and Methods 7
1. Materials 7
1.1 Cell lines 7
1.2 microRNA (miRNA) mimics 7
1.3 Reagents 8
2. Methods 8
2.1 RNA extraction 8
2.2 Real-time qRT-PCR 8
2.3 miRNA mimic transfection 9
2.4 Cell proliferation assay (Alamar blue assay) 9
2.5 Cell proliferation assay (Colony formation assay) 9
2.6 Transwell cell migration assay 10
2.7 Transwell cell invasion assay 10
2.8 GEO database analysis 10
2.9 Survival curve analysis 11
2.10 Target prediction analysis 11
2.11 Statistical analysis 11
2.12 Cell migration-TrackMate analysis 11
III. Result 13
1. The expression of candidate miRNAs in bladder cancer is associated with overall survival of patients 13
2. The expression of target genes in bladder cancer is associated with overall survival of patients 13
3. Candidate miRNAs cannot significantly regulate cell proliferation in bladder cancer
Cells 14
4. Candidate miRNAs regulate cell migration and invasion in bladder cancer cells 15
5. Candidate miRNAs regulate cell migration by TrackMate analysis 16
6. The Expression of candidate miRNAs in bladder cancer 16
7. Candidate miRNAs were regulated in bladder cancer cell lines 16
IV. Conclusion and discussion 18
1. Expression of candidate miRNAs and their target genes are correlated with overall survival of bladder cancer patients 18
2. Function of candidate miRNAs in bladder cancer cells 18
3. Expression of candidate miRNAs in patient serum and bladder cancer cell 18
4. Role of candidate miRNAs in bladder cancer 19
5. Investigation of miR-17-92 cluster 20
6. Future aspect 20
V. References 21

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指導教授

馬念涵(Nianhan Ma)

審核日期

2019-8-15

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