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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張逸良,楊泮池 | |
| dc.contributor.author | Hao-Cheng Kuo | en |
| dc.contributor.author | 郭皓程 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:08:04Z | - |
| dc.date.copyright | 2013-09-24 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17348 | - |
| dc.description.abstract | Pancreatic cancer is a lethal disease needing vigorous bio-medical research. We have established a pancreatic cancer cell line, KM cells, from a patient with poorly-differentiated ductal adenocarcinoma (PDAC). In order to dig out novel genetic alterations and potential treatment targets, genome-wide studies have been carried out. In the transcriptome study, the fusion of DCUN1D3 5’-UTR and GPRC5B coding region yields the highest read numbers that support the fusion event. The fusion is verified by PCR followed by Sanger sequencing at both transcript and genome levels. We demonstrate that small interfering/hairpin RNA knockdown of GPRC5B in KM cells changes cell morphology from mesenchymal-like to epithelia-like. Moreover, migration and invasion abilities are attenuated when GPRC5B expression is downregulated. These findings are supported by the RT-qPCR and Western Blot results in that the expression of epithelial-mesenchymal transition (EMT)-related markers, such as SNAI1, SLUG, Vimentin and N-cadherin, are suppressed while the expression of E-cadherin is increased at both transcript and protein levels when GPRC5B is knocked down. The results demonstrated in this study suggest that GPRC5B may play a certain role in PDAC EMT and thus metastases. The role of DCUN1D3 5’-UTR in the regulation of GPRC5B gene expression needs further investigation. This is the first study of GPRC5B in oncology field. Detailed mechanism and involved signaling pathways deserve further studies to clarify its molecular functions in cancer. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:08:04Z (GMT). No. of bitstreams: 1 ntu-102-R00444006-1.pdf: 3362113 bytes, checksum: 25cd345815daa2ab8e3aaf37da94ba99 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 口試委員審定書 I
謝辭 II 中文摘要 III Abstract IV Contents VI Chapter 1 Introduction 1 1.1 Pancreatic ductal adenocarcinoma 1 1.2 Cancer metastasis 2 1.3 DCUN1D3 4 1.4 GPRC5B 4 1.5 Study aims 5 Chapter 2 Materials and Methods 6 2.1 Cell lines and culture conditions 6 2.2 Polymerase Chain Reaction 6 2.3 SiRNA transfection by Lipofectamine 2000 7 2.4 Lentivirus infection 7 2.5 Boyden chamber invasion assay 8 2.6 Wound healing migration assay 9 2.7 Protein extraction 9 2.8 Bio-Rad Protein Assay 10 2.9 Western blot 10 2.10 Real-time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-qPCR) 11 2.11 Statistical Analyses 12 Chapter 3 Results 13 3.1 Identification and verification of GPRC5B-DCUN1D3 fusion 13 3.1.1 Identification of GPRC5B-DCUN1D3 fusion by transcriptome seq 13 3.1.2 Verification of the GPRC5B-DCUN1D3 fusion event at mRNA level 13 3.1.3 Verification of the GPRC5B-DCUN1D3 fusion event at gDNA level 14 3.2 GPRC5B knockdown suppresses epithelial-mesenchymal transition 15 3.2.1 GPRC5B knockdown by small interfering/hairpin RNAs results in cell morphological changes 15 3.2.2 Downregulation of GPRC5B suppresses migration and invasion ability 15 3.2.3 GPRC5B knockdown downregulates EMT-related gene expression at both mRNA and protein levels 16 Chapter 4 Discussions 18 Chapter 5 Figures and Tables 21 Figure 1. Visualization of RNA-seq reads supporting the GPRC5B-DCUN1D3 fusion junction 21 Figure 2. PCR and Sanger sequencing verification of GPRC5B-DCUN1D3 fusion. 22 Figure 3. Demonstration of GPRC5B-DCUN1D3 genomic rearrangment. 23 Figure 4. SiRNA knockdown of GPRC5B in KM cells results in cell morphological changes. 24 Figure 5. ShRNA knockdown of GPRC5B in K_M cells results in cell morphological changes. 25 Figure 6. ShRNA knockdown of GPRC5B in KM cells attenuates cell migration ability 26 Figure 7. ShRNA knockdown of GPRC5B in KM cells reduces cell invasion ability 27 Figure 8. GPRC5B knockdown downregulates EMT-related gene expression at both mRNA and protein levels 29 Table 1. Sequences of primers used for real-time quantitative polymerase chain reactions 30 References 31 | |
| dc.language.iso | en | |
| dc.title | GPRC5B在胰管腺癌中參與上皮-間葉的轉型過程 | zh_TW |
| dc.title | GPRC5B is involved in Epithelial-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳振都,田郁文 | |
| dc.subject.keyword | 胰管腺癌,上皮-間葉轉型,GPRC5B,Transcriptome sequencing,Fusion genes, | zh_TW |
| dc.subject.keyword | Pancreatic ductal adenocarcinoma,EMT,GPRC5B,Transcriptome sequencing,Fusion genes, | en |
| dc.relation.page | 33 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2013-08-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 病理學研究所 | zh_TW |
| 顯示於系所單位： | 病理學科所 | |
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