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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 俞松良 | |
| dc.contributor.author | Shih-Chun Hsu | en |
| dc.contributor.author | 徐詩淳 | zh_TW |
| dc.date.accessioned | 2021-06-16T06:52:29Z | - |
| dc.date.available | 2019-10-09 | |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-07-22 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57580 | - |
| dc.description.abstract | SPANX (Sperm protein associated with the nucleus, X-linked) 基因家族表現在精子生成的過程。在過去的研究中SPANX被認為是一種CTA (Cancer/Testis Antigen)，高度的表現在睪丸以及很多的癌症，像是膠質母細胞瘤、黑色素細胞瘤、睪丸生殖細胞瘤和惡性血液疾病等。然而，SPANX的功能以及牽涉在腫瘤進程中的機制卻仍然未知。我們先前的研究中顯示，SPANX gene family member A1 (SPANXA1) 在低侵襲性的肺癌細胞株CL1-0呈現高表現量，在高侵襲性細胞株CL1-5則表現較低。從體外細胞實驗中發現，穩定表現 SPANX1的CL1-5細胞株和陰性對照組株相比，其細胞侵襲的能力下降。為了進一步釐清SPANA1下游可能的傳遞訊息，我們使用基因表現微陣列晶片比較SPANXA1穩定表現細胞株與陰性對照組細胞株的基因表現差異，而實驗結果顯示在前十名的路徑中，有六個為表皮間葉細胞轉型過程(EMT)有關的訊息傳遞路徑，也發現細胞型態產生巨大的變化，從本來的間質型態變成表皮型態，呈現典型的反轉表皮間葉細胞轉型過程(MET)，此現象與其侵襲能力下降吻合。在SPANXA1穩定表現的細胞株中，我們使用免疫墨點法確定表皮型標誌蛋白E-cadherin表現量上升而間質型標誌蛋白vimentin表現量則下降。透過定量反轉錄聚合酶連鎖反應去驗證在個別路徑中重要的基因表現。在穩定表現SPNANXA1的細胞株中，調控表皮間葉細胞轉型過程的轉錄因子Slug有下降的趨勢，而在靜默SPANXA1的細胞株中，Slug有上升的趨勢，Slug在過去的研究中顯示其可以作用在E-cadherin的啟動子上進而抑制其轉錄。綜合上述實驗結果都顯示出SPANXA1表現在肺癌時，為表皮間葉轉型過程中一個重要的調控因子，這也顯示出SPANXA1在未來有潛力發展在標靶治療。 | zh_TW |
| dc.description.abstract | SPANX (Sperm protein associated with the nucleus, X-linked) gene family express throughout spermatogenesis in agreement with a potential role in sperm development. It also has been reported as a CTA (Cancer/Testis Antigen), which was highly expressed in testis and several cancers, such as glioblastoma, melanoma, testicular germ tumor and hematologic malignancies. Nevertheless, the function and molecular mechanism of SPANX involved in the cancer progression are greatly unknown. We found that the SPANX gene family member A1 (SPANXA1) was high-expressed in CL1-0, a lower invasive lung cancer cell line, and low-expressed in CL1-5, a higher invasive cell line. In vitro assays showed that the overexpression of SPANXA1 in CL1-5 cells suppresses invasive activity. To identify the underlying mechanism of SPANXA1, we preformed gene expression microarrays and the results indicated that EMT-related pathways are enriched in top ten pathways. Cell morphology is dramatically changed from mesenchymal-like to epithelial-like (MET). In presence of SPANXA1, the epithelial marker, E-cadherin, was up-regulated and the mesenchymal marker, Vimentin, was down-regulated. Quantitative RT-PCR assays were used to validate the differential expression of important genes in the identified pathways. The EMT regulating transcription factor, Slug, which acts a repressor for E-cadherin expression, was decreased in SPANXA1-overexpressed cells and increased in CL1-0 SPANXA1-slienced CL1-0 cells. These data suggest that SPANXA1 is a novel EMT modulator involved in lung cancer progression by mediating Slug expression and implies the therapeutic role of SPANXA1 in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T06:52:29Z (GMT). No. of bitstreams: 1 ntu-103-R01424023-1.pdf: 2753035 bytes, checksum: 4e44f09c56e899a232bda31dda1cc183 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
Acknowledgement II 中文摘要 III Abstract IV 1 Introduction 1 1.1 Lung Cancer 2 1.2 Metastasis 4 1.2.1 Initiation of metastasis 4 1.2.2 Dissemination 4 1.2.3 Survival in the circulation 5 1.2.4 Niche formation 6 1.2.5 Dormancy 6 1.2.6 Colonization 7 1.3 Epithelial-Mesenchymal transition 7 1.3.1 EMT in development 7 1.3.2 EMT in cancer progression 8 1.4 SPANXA1 9 1.4.1 SPANX gene family 9 1.4.2 SPANX in cancer research 10 1.5 Rationale 11 2 Materials and Methods 12 2.1 Cell culture 13 2.2 Plasmid construction and transfection 13 2.3 Lentiviral-based shRNA infection 14 2.4 Western blot analysis 15 2.5 In vitro matrigel invasion assay 16 2.6 Cell proliferation assay 16 2.7 Real-time Quantitative RT-PCR 17 2.8 Microarray pathway analysis 18 2.9 Immunofluorescence 19 2.10 Immunoprecipitation 19 2.11 Protein identification 20 2.11.1 Silver stain ( PlusOneTM Silver Staining Kit, Amersham Biosciences) 20 2.11.2 In-gel digestion 20 2.11.3 Analysis of LC-MS/MS 21 2.12 Statistical analysis 22 3 Results 23 3.1 The expression of SPANXA1 mRNA is higher in CL1-0 than in CL1-5 lung cancer cells. 24 3.2 Construction of SPANXA1 expression plasmid and establishment of SPANXA1-expressed transfectants. 24 3.3 SPANXA1 enhances the cell proliferation and down-regulates invasive ability in vitro. 25 3.4 Knockdown SPANXA1 in CL1-0 lung cancer cells up-regulates invasive ability. 26 3.5 Human Affymetrix Genechip and MetaCore pathway analysis. 26 3.6 SPANXA1 regulates genes which are enriched in EMT pathways. 27 3.7 SPANXA1 promotes MET in CL1-5 and localizes in the nucleus. 27 3.8 SPANXA1 regulates the expression of Slug. 28 3.9 The identification of SPANXA1-associated protein 29 4 Discussion 31 4.1 The variation between SPANX protein family 32 4.2 Slug may contribute to SPANXA1-suppressed metastasis 33 4.3 SPANXA1 may suppress EMT by forming a complex 34 4.4 Conclusion and future perspectives 36 5 Figures 38 Figure 1. Endogenous and Exogenous expression of SPANXA1. 39 Figure 2. The impact of SPANXA1 on invasion and cell growth in CL1-5 cells. 40 Figure 3. Selection of SPANXA1-expressed single clones. 41 Figure 4. Single clones of stably SPANXA1-expressed transfectants show largely decreased invasive ability versus the control. 42 Figure 5. Invasion-related phenotype of TRC Lentiviral-based shRNA knockdown in CL1-0 cells. 43 Figure 6. Fold expression changes of EMT-related genes. 44 Figure 7. EMT-related morphology and markers expression. 45 Figure 8. SPANXA1 regulates the expression of Slug. 46 Figure 9. Different bands of 12% SDS-PAGE between SPANXA1-V5 and IgG control by co-immunoprecipitation. 48 Figure 10. Different bands of 10% SDS-PAGE between SPANXA1-V5 and IgG control by co-immunoprecipitation. 49 Table 1. Enrichment pathways of SPANXA1 by Metacore. 50 Table 2. Expression validation of the top five up-regulated and down-regulate gene by Quantitative RT-PCR. 51 Table 3. The probable proteins of band 12-1 which were identified by Mascot Daemon software. 52 Table 4. The probable proteins of band 12-2 which were identified by Mascot Daemon software. 53 Table 5. The probable proteins of band 10-1 which were identified by Mascot Daemon software. 54 Table 6. The probable proteins of band 10-2 which were identified by Mascot Daemon software. 55 Table 7. The probable proteins of band 10-3 which were identified by Mascot Daemon software. 56 Table 8. The probable proteins of band 10-4 which were identified by Mascot Daemon software. 57 6 Appendices 58 Supplementary table 1. Design of the primers . 59 Supplementary figure 1. Construction map of pEF6/V5-His-TOPO-SPANXA1 61 Supplementary figure 2. mRNA expression of PKM2 and PRDX1 62 7 References 63 | |
| dc.language.iso | en | |
| dc.title | 新穎的控制因子SPANXA1調節肺癌的表皮間葉細胞轉型過程 | zh_TW |
| dc.title | A Novel Modulator, SPANXA1, Regulates Epithelial-Mesenchymal Transition in Lung Cancer | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李財坤,詹迺立,張正琪,蘇剛毅 | |
| dc.subject.keyword | 表皮間葉細胞轉型過程,癌轉移,微陣列基因晶片,Slug,抑癌基因, | zh_TW |
| dc.subject.keyword | EMT,Metastasis,Microarray,Slug,Tumor suppressor, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-07-22 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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