VHL剔除模型之轉錄體差異以及台灣透明細胞腎細胞癌族群之特定基因體變異之研究

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許鈞亮(Chun-Liang Hsu) 查詢紙本館藏

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系統生物與生物資訊研究所

論文名稱

VHL剔除模型之轉錄體差異以及台灣透明細胞腎細胞癌族群之特定基因體變異之研究
(The study of transcriptome differentiation in VHL knockout model and specific genomic variations in Taiwanese ccRCC population)

相關論文

★ 由基因微陣列分析發炎與腎臟細胞癌發生之機制	★ VHL基因突變在癌前期的組織發炎機制
★ VHL knockdown HK-2 cells induce macrophage endothelial extravasation	★ ITPR2, an ER calcium channel, regulates ER stress and inflammatory response in pre-cancerous kidney tubule cells
★ 透明腎臟細胞癌發生前期與組織發炎之關係研究	★ VHL與KIM-1的功能關係研究
★ 血管內皮細胞在腫瘤微環境中促進透明腎細胞癌形成之研究	★ Study of the Interaction between VHL/Vhlh Deficient Kidney Epithelial Cells and Macrophages—Relevance to the Development of Clear-Cell Renal Cell Carcinoma
★ 應用大腸桿菌與酵母菌蛋白質體晶片系統性分析抗菌肽及抗生素作用之目標蛋白質	★ Analysis of Gene Expression of Chronic Obstructive Pulmonary Disease and Chronic Kidney Disease to Illuminate Chronic Inflammation Associated with Tumor Microenvironment and Potential Treatment

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摘要(中)

透明細胞腎細胞癌佔據約６０％腎臟惡性腫瘤，其中７０％以上的透明細胞腎細胞
癌源自於VHL 腫瘤抑制基因的失能，VHL 基因的失能導致低氧誘導轉錄因子的累積，使
低氧誘導因子活化低氧反應基因，如：血管內皮生長因子，其能促進血管增生。低氧誘
導因子也能於低氧環境下調節代謝適應基因。先前的研究中，我們已經發現VHL 剔除腎
臟的差異基因表達與細胞增生、細胞分化、免疫反應以及細胞代謝有關。此外我們連結
發炎反應與VHL 失能誘導的內質網壓力的關係。然而，腎小管上皮細胞的基因表達特異
性仍屬於未知，我們也注意到一個鈣離子通道ITPR2 基因活性在透明細胞腎細胞癌中常
有增加現象，我們也懷疑其功能是否能誘導內質網壓力的惡化。因此，這篇研究中，我
們利用腎小管上皮細胞作為模型，利用RNA 定序來分析其轉錄體的變化，以及利用ＤＮ
Ａ定序分析基因體的突變。我們發現部份上調表現的基因，如：Cxcl12、Vegfa、Tgfa、
Cldn8、Cldn9 以及Vasp 能夠促進腫瘤前期的生成。另外，Egln3、Peg3os、Cd82、Scnn1b
以及Sulf1 卻能夠抑制腫瘤生成。有些下調的基因能促進腫瘤的形成，如：Adam9；調
節異常的代謝，如Cbs 以及Kcnma1；或是抑制腫瘤生長，如：Sptan1。這提供我們許多
有關腫瘤形成前轉錄體的轉變訊息。我們也發現台灣透明細胞腎細胞癌患者帶有VHL 突
變的比例與世界是一致的，且我們也找到ITPR2 單核甘酸多樣性能夠增強VHL 失能造成
的內質網壓力的反應。這提供我們ITPR2 與透明細胞腎細胞癌生成間的關係。

摘要(英)

Clear-cell renal cell carcinoma (ccRCC) constitutes 60% renal malignancy and up to 70%
of ccRCC cases result from loss function of the von Hippel-Lindau (VHL) tumor suppressor
gene. Loss-of-function of VHL gene causes accumulation of hypoxia inducible factor 1 alpha
(HIF1-α) transcription factor, then HIF1-α can activate hypoxia-responsive genes such as
vascular endothelial growth factor (VEGF) gene that promotes angiogenesis. HIF transcription
factor also regulates other genes involved in metabolic adaption to the hypoxia condition
(Warburg effect). In previous research, we have found that the differentially expressed genes in
Vhlh (mouse allele of the VHL gene) conditional knockout kidney were mostly involved in cell
proliferation, cell differentiation, immune response, and cell metabolism. In addition, we also
linked the inflammatory response with ER-stress induced by VHL inactivation. However, gene
expression patterns specific to renal tubular epithelial cells are still unclear. We are also
interesting in whether deterioration of ER-stress can be induced by the activity of a calcium
channel genes ITPR2, the amplification of which has been shown to correlate with the risk of
developing ccRCC. Therefore, in this research, we used the primary renal proximal tubular
epithelial cells as a model and analyzed the differential transcriptome by RNA sequencing, and
analyzed genomic mutation by DNA sequencing. We found that some upregulated genes such
as Cxcl12, Vegfa, Tgfa, Cldn8, Cldn9 and Vasp may promote cancer formation, but some genes
such as Egln3, Peg3os, Cd82, Scnn1b, and Sulf1 may inhibit tumorigenesis. The downregulated
genes may promote cancer formation such as Adam9, lead to aberrant metabolism such as Cbs
and Kcnma1, or inhibit cell growth such as Sptan1. The global gene expression profile of Vhlh
loss-of-function kidney tubule cells provided us with information about the transformation that
promote cancer formation. In addition, we found the proportion of VHL dysfunction in ccRCC
patients in Taiwan was consistent with prevalence in the world. And, we also found that single
nucleotide polymorphism for ITPR2 may enhance ER-stress response induced by VHL
dysfunction. This provided us with more information to link ITPR2 and ccRCC formation

關鍵字(中)

★ 透明細胞腎細胞癌

關鍵字(英)

★ VHL
★ ccRCC

論文目次

摘要 ..............................................................................i
Abstract ..........................................................................ii
誌謝 ..............................................................................iii
Table of Contents .................................................................iv
List of Figures ...................................................................v
List of Tables ....................................................................vii
Chapter 1 Introduction ............................................................1
1-1 Renal cell carcinoma and von Hippel-Lindau gene dysfunction ...................1
1-2 The prevalence of renal cell carcinoma in Taiwan ..............................3
1-2-1 Urothelial carcinoma and its prevalence .....................................3
1-2-2 The change of DNA sequence in ccRCC .........................................5
Chapter 2 Material and Methods ....................................................7
2-1 Animal ........................................................................7
2-2 Mouse renal tubule isolation ..................................................7
2-3 RNA extraction and RNA sequencing ............................................ 9
2-4 Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (QRT-PCR) .. 10
2-5 Patient samples ...............................................................12
2-6 DNA extraction ................................................................12
2-7 DNA sequencing and bioinformatics .............................................13
2-8 Statistical analysis ..........................................................13
Chapter 3 Results .................................................................14
3-1 Renal cell carcinoma and von Hippel-Lindau gene dysfunction ...................14
3-2 The prevalence of renal cell carcinoma in Taiwan ..............................24
Chapter 4 Conclusion and Discussion ...............................................30
Chapter 5 Reference ...............................................................43
Appendix ..........................................................................51

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指導教授

徐沺(Tien Hsu)

審核日期

2019-1-10

推文