泌尿道上皮癌相關 miRNAs 在膀胱癌幹細胞能力的 影響之研究

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姓名

馮于甄(Yu-Chen Fung) 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 miRNAs 在膀胱癌幹細胞能力的影響之研究
(Study of the effects of urothelial carcinoma- related miRNAs on bladder stem cell ability)

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至系統瀏覽論文 (2028-10-20以後開放)

摘要(中)

在人類泌尿道系統中，泌尿道上皮癌(Urothelial carcinoma, UC)是最常見的惡性腫瘤，約佔膀胱癌(Bladder cancer, BC)的 90%。患者經過化療或手術後會有高機率復發和轉移情況出現。根據文獻報導，癌症幹細胞(Cancer stem cell, CSC) 會在化療後聚集，因此導致治療失敗原因之一。最近也發現 miRNA 被認為是治療癌症的分子生物標誌物以及治療靶點，加上先前實驗室研究已證明，六種 UC 血漿 miRNAs(包括 miR-19b-1-5p、miR-150-5p、miR-155-5p、miR-210-3p、miR-378a- 3p、miR-636)可以區分 UC 和 non-UC 的血液透析患者，因此想要在本研究中，探討 UC-相關 miRNAs 影響在 BC 細胞腫瘤中的癌幹細胞功能。先前文獻發現， miR-19b-1-5p、miR-210-3p 和 miR-378a-3p 的過表達可減少 BC 細胞的增殖和遷移，但 miR-155-5p、miR-150-5p 和 miR-636 會促進其 BC 細胞表現。然而，我們結果表明，miR-19b-1-5p、miR-210-3p、miR-378a-3p 和 miR-155-5p 的過表達可以顯著性減少 J82 和 RT4 細胞的增殖和致瘤性。另一個結果表明，miR-150-5p 和 miR- 155-5p 的過表達可以顯著性降低 J82 和 RT4 細胞的遷移。此外，miR-493-3p、miR- 155-5p 和 miR-636 的過表達通過球體形成實驗會顯著性降低 J82 和 RT4 細胞的癌幹細胞能力。在西方墨點法中，透過 CSC 標記物(CD44 和 Nanog、Sox2)的表達，證明其表現在 J82 和 RT4 球體中顯著性下降。因此，我們進一步收集細胞球體，並進一步利用免疫螢光測定以研究 CSC 標記物的表現，加以證明球體經過 miRNAs 處理後會表達 Nanog。總之，我們的結果表明 UC-相關的 miRNAs 參與了 BC 的進展，可以被認為是 BC 的新治療選擇。

摘要(英)

Urothelial carcinoma (UC) is the most common malignancy type of bladder cancer (BC)
in the human urinary system, with about 90%. The patients had a high recurrence and
metastasis after the chemotherapy or surgery. It was reported that cancer stem cells
(CSC) are enriched after chemotherapy, leading to treatment failure. Recently, miRNAs
have been considered molecular biomarkers and therapeutic targets for the treatment of
cancers. Our previous study demonstrated that the expressions of six UC plasma
miRNAs - including miR-19b-1-5p, miR-150-5p, miR-155-5p, miR-210-3p, miR-
378a-3p, miR-636 could distinguish UC and non-UC patients with hemodialysis. In this
study, we investigated the biological functions of these UC-related miRNAs in the
tumorigenesis of BC cells. It had reported that miR-19b-1-5p, miR-210-3p, or miR-
378a-3p expressions could reduce BC cell proliferation and migration, but miR-155-5p,
miR-150-5p or miR-636 promoted BC cell proliferation and migration. However, our
results showed that overexpression of not only miR-19b-1-5p, miR-210-3p, or miR-
378a-3p but also miR-155-5p could reduce the proliferation and anchorage-
independent growth of J82 and RT4 cells significantly. Another result showed that
overexpression of miR-150-5p or miR-155-5p could facilitate the migration of J82 and
RT4 cells significantly. In addition, overexpression of miR-493-3p, miR-155-5p, or
miR-636 decreased the stem cell ability of J82 and RT4 cells by sphere formation assay.
Consistently, the CSC markers (CD44, Nanog, and Sox2) expression significantly
declined in the J82 and RT4 spheres. Thus, we further dissected the cultured urothelial
stem cells from sphere formation and performed an immunofluorescence assay to
investigate the expression of CSC markers. After miRNA treatment, we demonstrate that these spheres would express the stem cell markers NANOG. In summary, our results suggested that UC-related miRNAs were involved in the progression of BC and could be considered novel therapeutic options for BC.

關鍵字(中)

★ 泌尿道上皮癌
★ 癌症幹細胞
★ 微型核糖核酸

關鍵字(英)

★ Urothelial Carcinoma
★ Cancer Stem Cell
★ microRNA

論文目次

中文摘要 I
ABSTRACTII
圖目錄..VIII
ABBREVIATION LISTIX
一介紹（INTRODUCTION）.1
1. 膀胱癌（BLADDER CANCER） .1
1.1 泌尿道上皮癌（Urothelial carcinoma）.1
1.2 診斷與治療（Diagnosis and Treatment）..2
2. 癌症幹細胞（CANCER STEM CELL）.3
2.1 癌症中的癌症幹細胞（Cancer Stem Cells in Cancer）.3
2.2 調控膀胱癌的途徑（Pathways Regulating of Bladder Cancer）.4
2.3 膀胱癌的潛在治療靶點（Potential therapeutic target for bladder cancer） .4
3. 微型核糖核酸（MICRORNA）..5
3.1 miRNA 的發現（Discovery of miRNAs） 5
3.2 miRNA 的生成與調控（Biogenesis and regulation of miRNA） .6
3.3 miRNA 的失調與應用（miRNA dysregulation and application in cancer）.7
4. 研究目的（RESEARCH PURPOSE）..8
4.1 探討 UC 相關微型 RNA 在膀胱癌中的作用..8
（Exploring the role of UC-associated microRNAs in bladder cancer）..8
二實驗材料與方法（MATERIALS AND METHODS）.. 10
1. 實驗材料（MATERIAL） .. 10
1.1 細胞株（Cell lines）.. 10
1-2 微型核糖核酸模擬物（miRNA mimics）. 10
1-3 試劑（Reagents）.. 10
1-4 抗體（Antibodies） . 10
2. 實驗方法（METHOD） 11
2-1 miRNA 模擬物轉染（miRNA mimic transfection）.. 11
2-2 細胞群落形成實驗（Colony formation） 11
2-3 細胞不依賴性生長（Anchorage-independent growth） 11
2-4 細胞爬行實驗（Cell migration assay）. 11
2-5 細胞球體形成實驗（Sphere formation assay） 11
2-6 蛋白質萃取製備（Preparation of protein extraction）.. 12
2-7 西方墨點法（Western blot）. 12
2-8 免疫螢光染色（Immunofluorescence staining）.. 13
2-9 統計 (Statistics) .. 13
三實驗結果（RESULTS）.. 14
1. UC-RELATED MIRNAS 對於膀胱癌細胞的生長和存活的影響。 14
2. UC-RELATED MIRNAS 對於膀胱癌細胞的致瘤性和轉化的影響。. 14
3. UC-RELATED MIRNAS 對於膀胱癌細胞的遷移的影響。 .. 15
4. UC-RELATED MIRNAS 對於膀胱癌細胞形成球體的影響。. 16
5. UC-RELATED MIRNAS 調節膀胱癌細胞中的 BC 幹細胞能力。 . 16
四結論（CONCLUSION） 18
五討論（DISCUSSION） 19
1. UC-RELATED MIRNAS 對於癌細胞的生長、存活、致瘤性和遷移的影響.. 19
2. UC-RELATED MIRNAS 對於癌症幹細胞的影響. 20
3. 探討 MIR-493-3P 在膀胱癌中扮演的角色.. 21
4. 探討 UC-RELATED MIRNAS 可能會參與的路徑進而影響膀胱癌的發生和復發. 21
5. 未來展望.. 22
六參考文獻（REFERENCE）.. 23

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指導教授

馬念涵(Nianhan Ma)

審核日期

2023-10-20

推文