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研究生:吳華席
研究生(外文):Hua-Hsi Wu
論文名稱:卵巢癌微小核醣核酸表現譜與鉑類抗葯性之關聯
論文名稱(外文):MicroRNA profiling is associated with platinum-resistance in ovarian cancer
指導教授:袁九重袁九重引用關係林文昌林文昌引用關係
指導教授(外文):Chiou-Chung YuanWen-Chang Lin
學位類別:博士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:83
中文關鍵詞:微小核醣核酸表現譜鉑類抗葯性卵巢癌
外文關鍵詞:MicroRNA profilingplatinum resisatnceovarian cancer
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卵巢癌是最易致死的婦癌。其標準療法是先減積手術後輔以合併鉑類與太平洋紫杉醇的化學治療。此項療程一開始幾乎都可使疾病得到控制,但最後多不免復發並平均在初次後發的兩年後因抗葯性產生而死亡。目前再復發選擇化療葯的種類是依據「無鉑期」。若無鉑期大於6個月,就可再次使用包含鉑類的化學治療。一旦無鉑期越長,則對第2次化療反應會越好。因此有一些學者便建議以各種人為方法來延長無鉑期,從而使病人重新得到或加強對鉑類治療的反應。但此一假設仍缺乏強有力的証据來支持它。
微小核醣核酸是一群富含於多細胞生物,長度約為19-22個核苷酸的核醣核酸。它們不會製造蛋白質,但卻能經由許多種機轉來影響基因的表現。目前已知存在於人類的微小核醣核酸已有2500餘種。微小核醣核酸表現譜於許多種癌症中已引起相當注意、近年來他在正常組織與癌組織間已被證明會有不同的表現。有一些研究顯示藉由表現譜的不同表現,可幫助癌細胞的分類和分期,其至還可預知預後並協助選擇治療方式。
為了證實上述的假設並試著找出其機轉,我們使用卵巢癌細胞株給予不同 的化療方式以模擬不同的化療臨床處理狀況,除了觀察細胞對藥物反應的敏感度,同時還測量378 種微小核醣核酸。我們的實驗顯示即使只有短暫的無鉑期於體外培養的卵巢癌細胞仍可使恢復部份對順鉑的敏感性。我們證明在不同治療方法下會有其獨特的微小核醣核酸表現譜。同時亦發現有一群微小核醣核酸與順鉑敏感性的部份恢復有關。
我們證明延長無鉑期可以使卵巢癌細胞重新得到或加強對鉑類治療的反應,而微小核醣核酸表現譜對研究順鉑敏感性恢復的機轉可提供重要的幫忙。

Ovarian cancer is the most lethal cause among gynecological malignancies. Most of them are diagnosed at advanced stage. The standard treatment for advanced ovarian cancer is debulking surgery with adjuvant platinum-paclitaxel combination chemotherapy. In spite of the high response rate of initial treatment, the majority will relapse and eventually die due to the development of chemoresistance. The probability of response to retreatment with platinum depends on the platinum-free interval (PFI). It may be possible to increase response rates when re-introducing platinum by extending the PFI using non-platinum regimens. However, this hypothesis still lacks solid proof and remains to be debated.
MicroRNA is a group of small non-coding RNAs of 19-22 nucleotides in length that are broadly abundant in all multicellular eukaryotic cells. More than 2500 mature miRNAs have been identified in human. The miRNA profiling can classify stage, subtype, responsibility to treatments and prognosis in several hematopoietic and solid tumors.
Our results showed that extension of PFI could partially restore the platinum sensitivity. Six miRNAs located in two neighboring areas were identified to be correlated with the recovery of cisplatin sensitivity.
Our data implied that the ovarian cancer cells can partially retain their platinum sensitivity after platinum-free interval, even a short interval, in vitro. MicroRNA regulation may play a role in recovery of platinum-resistance after platinum-free interval. The miRNAs with significant fold-change may be a biomarker to predict the response to platinum re-introduction.
Table of Contents
English Abstract ……………………………………………………………… I
Chinese Abstract …………………………………………………………….. III
List of Abbreviations ………………………………………………………... IV
CHAPTER 1 Introduction ………………………………………………….. 1
1.1 MicroRNA ……………………………………………………………… 1
1.1.1 Biogenesis of microRNAs ……………………………………….. 1
1.1.2 Mechanisms of action of miRNAs …………………………….... 3
1.1.3 MiRNAs and cancer ……………………………………………….. 4
1.1.4 MiRNAs and epithelial ovarian cancers ……………………….. 5
1.1.5 MiRNA profiling …………………………………………………….. 6
1.1.6 MiRNAs in chemotherapy ………………………………………... 7
1.2 Epithelial ovarian cancer …………………………………………… 8
1.2.1 Epithelial ovarian cancer and cancer stem cells ………….... 10
1.2.2 Circulating biomarkers for ovarian cancers …………………… 11
1.2.3 Chemotherapy for epithelial ovarian cancer ………………… 12
1.2.4 Chemotherapy at relapse …………………………………………. 13
1.2.5 The clinical importance of platinum-free interval and definition of platinum sensitivity …………………………………………….. 15
1.2.6 Clinical significance of extending the platinum-free interval.. 16
1.3 Goal of our studies …………………………………………………… 17
CHAPTER 2 Materials and Methods ……………………………………… 19
2.1.1 Selection of Cell line ………………………………………………..19
2.1.2 Determination of the optimal experimental drug concentrations by cell viability assay ……………………………………………. 19
2.1.3 The TOV-21G cells were classified to four groups according to different treatments ………………………………………………. 20
2.1.4 RNA extraction and quality control test ……………………… 20
2.1.5 MiRNAs Microarray Analysis …………………………………… 21
2.1.6 Reverse transcription of miRNAs ……………………………… 21
2.1.7 Real-time PCR for miRNAs ……………………………………… 22
2.1.8 Data analysis ………………………………………………………. 22
2.1.9 Statistical analysis ……………………………………………….. 23
2.2.1 Cell Morphology of TOV-21G seeded in normal plates or bacterial petridishes …………………………………………… 23
2.2.2 RNA extraction and Real-time quantitation RT-PCR for CSCs & EMT markers expression profile ………………………………. 24
2.2.3 Paclitaxel treatment of TOV-21G cells seeded in normal plates or bacterial petridishes ……………………………………..…... 24
2.2.4 Side population (SP) cells analysis by Flow-cytometry …… 25
CHAPTER 3 Results ………………………………………………………… 26
3.1 TOV-21G cell line was selected as the experimental cells …… 26
3.2 Cell viability after re-introduction of cisplatin …………………. 26
3.3 MiRNAs expression profiling ……………………………………… 27
3.4 The potential miRNAs associated with the recovery of platinum-sensitivity ……………………………………………….. 27
3.5 The morphologic change of non-anchoraging ovarian cancer cells …………………………………………………………………… 28
3.6 Enhancement of stem cell and EMT markers expression of non-anchoraging ovarian cancer cells …………………………. 30
3.7 The non-anchoraging ovarian cancer cells with enhanced CSCs markers were not side-population cells ……………………… 31
CHAPTER 4 Discussion ........................................................................ 33
CHAPTER 5 Conclusion ........................................................................ 38
CHAPTER 6 Perspectives ..................................................................... 39
References …………………………………………………………………… 40
Tables and Figures …………………………………………………………… 60
Publications …………………………………………………………………… 82

Figures
Figure 1. TOV-21G cells had moderate inherent resistance to various chemotherapy …………………………………………………. 61
Figure 2. Determination of the optimal experimental drug concentrations by cell viability assay ……………………. 61
Figure 3. The cell survival rates after re-introduction of differential cisplatin doses in each group with different treatment protocol ………………………………………………………. 62
Figure 4. Distinct miRNA expression profiling after re-introduction of differential cisplatin doses in each group with different treatment protocol by cluster ………………………………. 63
Figure 5. The change of ΔCt values of the potential miRNAs associated with recovery of platinum sensitivity ………. 64
Figure 6. The morphology of TOV-21G grown in different culture conditions ……………………………………………………… 65
Figure 7. Electrophoresis grams of stem cell biomarker gene expression profile of TOV-21G cells………………………… 66

Figure 8. Side population analysis by flow cytometry …………… 67


Tables
Table 1. The role of microRNA in ovarian cancer …………………… 68
Table 2. The sequence list of the stem cell markers of real-time PCR primers ………………………………………………………….. 71
Table 3. The sequence list of EMT markers of real-time PCR primers …………………………………………………………. 72
Table 4. Quantification of Total RNA using NanoDrop ND-1000 …. 73
Table 5. p-values of comparison of intergroup cell viability by paired t-test ………………………………………………………………. 74
Table 6. Differentially expressed miRNAs associated with different chemotherapy …………………………………………………. 75
Table 7. Stem cell biomarker gene expression profile of TOV-21G cells ………………………………………………………………. 76
Table 8. The EMT marker expression profile of TOV-21G ………… 78
Table 9. Stem cell biomarker gene expression profile of TOV-21G cells with paclitaxel treatment ……………………………………….. 79
Table 10. Characteristics of the miRNAs associated with the loss of platinum resistance after platinum-free treatment ……… 80








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