臺灣博碩士論文加值系統

在卵巢癌中，TGF-β訊息傳遞扮演著非常重要的角色。失控的TGF-β訊息傳遞除了會導致卵巢癌的發生也會使癌症幹細胞的存活。在本篇研究中，我們利用ChIP-chip和expression array找出和TGF-β/SMAD4相關基因：FBXO32和ABCA1，並研究其跟卵巢癌的相關性。在第一部分研究中，我們發現在正常卵巢細胞中FBXO32為正常表現，但是在卵巢癌細胞株中卻被抑制。近一步研究顯示，FBXO32表現異常是因為在啟動子產生甲基化現象，所以利用表基因藥物可以減少甲基化情形以及讓FBXO32重新表現。利用FBXO32甲基化位置來分析病人檢體，發現有FBXO32的甲基化，卵巢癌病人呈現較高的復發機率(29.3%; P<0.05)。在卵巢癌細胞株中重新表現FBXO32可以有效誘導細胞凋亡，抑制癌細胞生長，並且降低癌細胞對cisplatin的抗性。再第二部份研究中，我們從mDIP-Chip中發現ABCA1在A2780和CP70癌細胞株中有甲基化現象，以及從expression data中也可以發現ABCA1表現被抑制。更進一步研究顯示，ABCA1的不表現是因為啟動子部位的甲基化。接著分析ABCA1甲基化跟病人檢體的關係，發現在高stage跟高grade病患中，ABCA1甲基化情形也比較高(P=0.0169 vs. P=0.0024)。而且ABCA1甲基化較高的病患，也較易死亡(P=0.016)。在卵巢癌細胞中，FBXO32和ABCA1皆被DNA甲基化所抑制表現，且產生甲基化的病患都有不佳的癒後表現。

The Dysregulation of TGF-β signaling plays a key role in ovarian carcinogenesis and maintaining cancer stem cell properties. In this study, we utilized previous ChIP-chip, mDIP-chip, and expression array data to identify TGF-β/SMAD4 relative genes: FBXO32 and ABCA1. In the first part of study, we found that expression of FBXO32 was observed in normal ovarian surface epithelium but not in ovarian cancer cell lines. FBXO32 methylation was seen in ovarian cancer cell lines, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines, suggesting that epigenetic modifications regulate the expression of this gene in ovarian cancer. In advanced stage ovarian tumors, significant (29.3%; p<0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression free survival was significant (Kaplan-Meier, p<0.05). Re-expression of FBXO32 markedly reduced proliferation of ovarian cancer line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In the second part of the study, we identified ABCA1 by mDIP-Chip which was methylated in ovarian cancer cell line, A2780 and CP70. ABCA1 was expressed not only in IOSE, but in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines. In A2780 and CP70 ovarian cancer cell line, ABCA1 was down-regulated and was associated with promoter hypermethylation as demonstrated by bisulfite pyro-sequencing. Analysis of ABCA1 methylation in 8 normal OSE and 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation is associated with high stage and high grade (p=0.0169 vs. p=0.0024). Importantly, patients with higher methylation of ABCA1 have shorter progression free survival (p=0.09) and overall survival (p=0.016). In conclusion, both of FBXO32 and ABCA1 were repressed by DNA methylation in ovarian cancer, and hypermethylation of them was associated with poor prognosis in cancer patients.

致謝 - 1 -
摘要 - 2 -
Abstract - 3 -
Background review 6
Ovarian cancer 6
The TGF-β signaling pathway 8
Epigenetic modifications 9
Aberrant TGF-β signaling and epigenetic alteration in ovarian cancer 12
Hypothesis and Objective of study 13
PART I: Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer 14
Material and Methods 14
Patient samples 14
Cell culture and epigenetic treatment 14
DNA extraction 14
Bisulfite conversion and Combined Bisulfite Restriction Analysis (COBRA) 15
Methylation specific PCR (MSP) and real time quantitative methylation-specific PCR (qMSP) 16
RNA extraction, reverse transcription and quantitative reverse transcription-PCR 17
Plasmid construction 18
Transfection and colony formation assay 18
Cell proliferation assay 19
Flow cytometry analysis 19
In vivo tumorigenicity assay 20
Immunohistochemistry 20
Statistical analysis 21
Results 22
PART II: Hypermethylation of a TGF- target, ABCA1 is associated with poor prognosis in ovarian cancer patients 27
Material and Methods 27
Patient samples 27
Cell culture 27
DNA extraction, RNA extraction and reverse transcription 27
Quantitative reverse transcription-PCR 27
Bisulfite conversion and pyro-sequencing 28
Statistical analysis 28
Results 30
Conclusion and Discussion 32
Figure and Table 38
Fig 1 38
Fig 2 40
Fig 3 41
Fig 4 43
Fig 5 44
Fig 6 46
Fig 7 50
Fig 8 52
Fig 9 55
Fig 10 58
Fig 11 59
Fig 12 61
Fig 13 64
Table 1. Summary of clinico-pathological data of 96 ovarian cancer patients 65
Table 2 Primer sequences used in the study 66
Table 3. Effect of FBXO32 on cell cycle distribution of HeyC2 cells 67
Table 4. Association between methylation of FBXO32 and clinicopathological features of 96 ovarian cancer patients 68
Table 5. Multivariate analysis of survival by Cox proportional hazards model 69
Table 6. Summary of clinico-pathological data of 76 ovarian cancer patients 70
Table 7. Association between methylation of ABCA1 and clinicopathological features of 76 ovarian cancer patients 71
Table 8. Univariable analysis of survival by Cox proportional hazards model 72
Table 10 Media Formulations of ovarian cancer cell lines 73
Appendix 74
Original data methylation of FBXO32 and clinical pathological parameters in ovarian cancer patient samples 74
Original data: methylation of ABCA1 and clinical pathological parameters in ovarian cancer patient samples 78
A: pyro-sequencing data of ABCA1 79
Publications 87
References 88

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