利用MAPK訊息傳導路徑相關的miRNAs來治療BRAF抑制劑的抗藥性在黑色素瘤細胞中之研究

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姓名

蔡仁傑(Jen-Chieh Tsai) 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

利用MAPK訊息傳導路徑相關的miRNAs來治療BRAF抑制劑的抗藥性在黑色素瘤細胞中之研究
(Study of MAPK signaling pathway-related miRNAs in resistance to BRAF inhibitor in melanoma cells)

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摘要(中)

MAPK/ERK訊息傳導路徑的活化在黑色素瘤中扮演了很重要的角色，在之前的研究中大約有50%的黑色素瘤包含BRAF基因突變，其中80%是BRAFV600E的取代突變，這個突變持續性地誘導MAPK/ERK訊息傳導路徑的活化也造成癌症的惡性表現型。標靶藥物PLX4032是一個強效的小分子抑制劑，抑制BRAFV600E突變蛋白質來治療黑色素瘤，在第三期的病人中，經過PLX4032治療後至少有48%的人有效，然而這個治療受限於有些病人在治療七個月後迅速產生抗藥性。我們建立了對PLX4032產生抗藥性的細胞株，我們發現黑色素瘤對PLX4032產生抗藥性是透過過度活化MAPK/ERK跟PI3K/AKT訊息傳導路徑，微型RNA是一個很有潛力的治療藥物，因為它可以一次抑制多個訊息RNA跟抑制多個致癌相關訊息傳導路徑。我們利用先前微陣列晶片實驗的結果和GEO資料庫篩選出與MAPK相關的微型RNA，實驗結果顯示出分別的過度表現4個微型RNA可以有效的抑制細胞增生跟細胞爬行，以上證據說明了微型RNA可能是個很有潛力的治療用來對抗產生PLX4032抗藥性的黑色素瘤。

摘要(英)

The activity of mitogen-activated protein kinase (MAPK/ERK) signaling plays an essential role in melanoma. It has been reported that approximately 50% of melanoma harbors activating BRAF mutations (over 80% BRAFV600E). The mutation constitutively induces high activity in MAPK/ERK signaling pathway and causes malignant phenotypes of cancers. PLX4032 is a selective and potent small molecule inhibitor of the V600E mutant form the BRAF protein used in the treatment of melanoma. In a phase III trial in patients, PLX4032 treatment resulted in a 48% response. However, this therapeutic success is limited by the rapid emergence of drug resistance after an average 7 months. We constructed a cell model of resistance to PLX4032 progressed by treatment of BRAFV600E melanoma cells with the BRAF inhibitor PLX4032. We demonstrated that MAPK/ERK and PI3K/AKT signaling pathways activity were reactivate in PLX4032-resistant melanoma cells. miRNA are excellent therapeutic candidates because of their ability to repress several mRNA or multiple oncogenic pathways at once. We screen out MAPK-related candidate miRNAs from the prior microarray results and GEO data analysis. Our data showed that overexpression of four miRNAs individually could decrease cell proliferation, anchorage-independent growth and migration. These results suggest that miRNAs could be a potent therapeutic candidate for counteracting treatment resistance of PLX4032 in melanoma.

關鍵字(中)

★ MAPK訊息傳導路徑
★ 微型RNA
★ 抗藥性黑色素瘤

關鍵字(英)

論文目次

目錄
摘要 i
Abstract ii
致謝 iii
目錄 v
List of figures vii
List of tables vii
Abbreviation list viii
I. Introduction 1
1. Melanoma 1
1.1 Genetics of Melanoma 1
1.2 Dysregulation pathway in Melanoma 2
1.3 Melanoma therapy 4
2. Drug resistance in Melanoma 5
2.1 Mechanism of resistance to BRAF inhibitor in Melanoma 5
3. miRNA 6
3.1 Biogenesis and mechanism of miRNA 6
3.2 miRNA dysregulation in cancer 7
3.3 miRNA application in cancer 8
4. Purpose and significance 9
4.1 To investigate the mechanism of miRNAs involved in overcoming the PLX4032-resistant melanoma 9
II. Materials and Methods 10
1. Materials 10
1.1 Cell lines 10
1.2 microRNA mimics 10
1.3 Drugs and Reagents 10
1.4 Antibodies 11
2. Methods 11
2.1 microRNA transfection 11
2.2 Preparation of protein extraction 11
2.3 Western blot analysis 12
2.4 Cell proliferation assay (Alamar blue assay) 12
2.5 Cell proliferation assay (Colony formation assay) 13
2.6 Soft agar assay 13
2.7 Transwell assay 14
2.8 Wound healing assay 14
2.9 Caspase3/7 assay 14
2.10 GEO database 15
2.11 Statistical analysis 15
III. Result 16
1. Expression of miR-524-5p, miR-596, miR-567, miR-518f-5p and miR-518d-5p is decrease in melanoma 16
2. Establishment of PLX4032-resistant Melanoma cell lines 16
3. PLX4032 acquired resistance display differential MAPK signaling pathway reactivation 17
4. MAPK signaling pathway-related miRNAs decrease cell proliferation and increase cell apoptosis in PLX4032-resistant SK-Mel-19 cells 18
5. MAPK signaling pathway-related miRNAs repress cell anchorage-independent growth in PLX4032-resistant SK-Mel-19 cells 19
6. MAPK signaling pathway-related miRNAs inhibit cell migration in PLX4032-resistant SK-Mel-19 cells 19
7. MAPK signaling pathway-related miRNAs down-regulate MAPK signaling pathway activity 20
8. MAPK signaling pathway-related miRNAs decrease PI3K/AKT signaling pathway activity 20
IV. Conclusion and discussion 22
1. Candidate miRNAs suppress MAPK or PI3K/AKT signaling pathway in PLX4032-resistant melanoma cells 22
2. Study of miRNAs involved in melanoma resistant to target therapy 23
3. Future aspect 23
V. References 25

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指導教授

馬念涵(Nian-Han Ma)

審核日期

2016-11-4

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