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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 顏伯勳(Bo-Shiun Yan) | |
| dc.contributor.author | You-Pen Chiu | en |
| dc.contributor.author | 邱有朋 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:07:31Z | - |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-18 | |
| dc.identifier.citation | References
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Oncogenic ras Provokes Premature Cell Senescence Associated with Accumulation of p53 and p16INK4a. Cell 88, 593–602. Moiseeva, O., Mallette, F. A., Mukhopadhyay, U. K., Moores, A., and Ferbeyre, G. (2006). DNA damage signaling and p53-dependent senescence after prolonged beta-interferon stimulation. Molecular Biology of The Cell 17, 1583-1592. Rufini, A., Tucci, P., Celardo, I., and Melino, G. (2013). Senescence and aging: the critical roles of p53. Oncogene 32, 5129-5143. Sato, M., Larsen, J. E., Lee, W., Sun, H., Shames, D. S., Dalvi, M. P., Ramirez, R. D., Tang, H., DiMaio, J. M., Gao, B., et al. (2013). Human lung epithelial cells progressed to malignancy through specific oncogenic manipulations. Molecular Cancer Research 11, 638-650. Sebastian, C., Zwaans, B. M., Silberman, D. M., Gymrek, M., Goren, A., Zhong, L., Ram, O., Truelove, J., Guimaraes, A. R., Toiber, D., et al. (2012). The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism. Cell 151, 1185-1199. Triulzi, T., Tagliabue, E., Balsari, A., and Casalini, P. (2013). LCTS1 expression in tumor cells and implications for cancer progression. Journal of Cellular Physiology 228, 30-35. Wang, L., Liu, R., Li, W., Chen, C., Katoh, H., Chen, G. Y., McNally, B., Lin, L., Zhou, P., Zuo, T., et al. (2009). Somatic single hits inactivate the X-linked tumor suppressorLCTS1 in the prostate. Cancer Cell 16, 336-346. Zuo, T., Liu, R., Zhang, H., Chang, X., Liu, Y., Wang, L., Zheng, P., and Liu, Y. (2007a). LCTS1 is a novel transcriptional repressor for the breast cancer oncogene SKP2. The Journal of Clinical Investigation 117, 3765-3773. Zuo, T., Wang, L., Morrison, C., Chang, X., Zhang, H., Li, W., Liu, Y., Wang, Y., Liu, X., Chan, M. W., et al. (2007b). LCTS1 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene. Cell 129, 1275-1286. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18483 | - |
| dc.description.abstract | 肺癌是由於肺組織中不受控制的細胞增生所引發的疾病。基於早期診斷的困難度以及不良預後的結果，肺癌平均的五年後存活率約只有15%，且每年造成約一百三十萬人死亡。LCTS1是一個轉錄因子（transcriptional factor）。因為LCTS1亦在肺細胞中表現，且在本實驗室先前的研究中，LCTS1的過度表現抑制了肺癌細胞株的發展（progression）。因此我們推測LCTS1在肺癌中依然是個腫瘤抑制者。為研究LCTS1在肺癌所扮演的角色，我們在無突變的正常肺細胞株中造成LCTS1的功能喪失來探討LCTS1的功能。我們在人類肺纖維細胞株WI-38中引進調控LCTS1之小髮夾核醣核酸（short hairpin RNA）慢病毒，造成LCTS1表現降低。LCTS1表現降低並無造成惡性轉化（malignant transformation）; 但替代地，我們觀察到細胞型態上的改變，並經由細胞衰老β-半乳糖苷酶染色（senescence-associated β-galactosidase staining）證明其與細胞老化相關的生長制止（growth arrest）現象有關。本研究亦發現p53蛋白的總量和磷酸化的增加，推論細胞的衰老表徵是經由p53受去氧核醣核酸損壞反應的調控所引起。此外，衰老細胞內下游蛋白p21的表現量與RB蛋白的磷酸化亦受到影響。我們的發現顯示LCTS1是一個有潛力的腫瘤抑制者，其功能喪失會造成去氧核醣核酸損壞的壓力並因此導致細胞的衰老。 | zh_TW |
| dc.description.abstract | Lung cancer is characterized as a disease caused by uncontrolled cell growth that occurs in lung tissue. The overall five-year survival rate for lung adenocarcinoma is probably 15% due to the difficulty of early diagnosis and the worse outcome of therapy, and the disease is responsible for about 1.3 million deathsannually.LCTS1(Lung Cancer Tumor Suppressor 1) is a transcriptional factor that is also expressed in lung, and the previous study in our lab demonstrated that overexpression of LCTS1 inhibits the progression of lung cancer cell lines. Therefore, we speculate that LCTS1 also functions as a tumor suppressor in the lung. To investigatethe roles of LCTS1 in lung cancer, we modeled loss of function of LCTS1 in the normal lung cell line. We knocked down LCTS1 by lentivirus-mediated introduction of LCTS1shRNA in WI-38, a human lung fibroblast cell line, and demonstrated that LCTS1 knockdown didn’t cause malignant transformation and, instead, the cell with LCTS1 knockdown showed morphologic change and growth arrest corresponded with cellular senescence that was proved by senescence associated β-galactosidase staining. Moreover, thelevels of expression and phosphorylation of p53 were up-regulated, suggesting that senescent phonotype was mediated by p53, which is induced by DNA damage response. The studies also revealed that the levels of p21expression as well as RB phosphorylation were changed in the senescent cells. Our findings suggest that LCTS1is a potential tumor suppressor and loss of which contributes to oncogenic stress thereby cells become senescent. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:07:31Z (GMT). No. of bitstreams: 1 ntu-103-R00442029-1.pdf: 1670344 bytes, checksum: a9a84adae4da5d083ed048460c0d0a5f (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | Chapter 1 Introduction ...............................................................................................8
1.1 Lung cancer ...............................................................................................8 1.2 LCTS1 .......................................................................................................9 1.3 Senescence...............................................................................................10 1.3.1 Replicative senescence ................................................................ 10 1.3.2 Premature senescence .................................................................. 11 1.3.3 Oncogene-induced senescence (OIS) .......................................... 12 1.4 Evidence and biomarkers of cellular senescence: ...................................13 1.4.1 Cell cycle arrest: .......................................................................... 13 1.4.2 Senescence-associated β-gal activity: .......................................... 13 1.4.3 Augmented tumor suppressor genes expression:......................... 13 1.4.4 Senescence associated heterochromatic foci (SAHF) ................. 14 1.4.5 Morphological transformation ..................................................... 15 1.4.6 Senescence-associated secretory phenotype ................................ 15 Chapter 2 Materials and Methods ............................................................................16 2.1 Cell cultural condition and drug treatment ..............................................16 2.2 ShRNA clones and plasmid construction ................................................17 2.3 Dual promoter Tet-on expression system................................................17 2.4 Colony formation assay...........................................................................18 2.5 Lentiviral production and infection .........................................................18 2.6 Senescence associated β-galactosidase staining......................................19 2.7 RNA extraction and quantitative reverse transcription and polymerase chain reaction (Q-RT-PCR)..............................................................................19 2.8 Western blot analysis:..............................................................................20 2.8.1 Cell lysate preparation ................................................................. 20 2.8.2 BCA protein quantification assay ................................................ 20 2.8.3 Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis .... 20 2.9 MTT proliferation assay ..........................................................................21 2.10 Soft agar colony formation assay ............................................................22 Chapter 3 Results .....................................................................................................23 Chapter 4 Discussion and Conclusion......................................................................28 Chapter 5 Figures .....................................................................................................31 Chapter 6 Plasmid maps and Tables.........................................................................44 Figure Contents Figure 1. The schematic diagram of the major LCTS1 isoforms and LCTS1 shRNA binding sites ......................................................................................................... 31 Figure 2. The LCTS1 mRNAs in WI-38 fibroblasts were successfully knocked down by prolonged shRNA expression .............................................................................. 32 Figure 3. LCTS1 knockdown promoted cell morphologic change .............................. 33 Figure 4. LCTS1 knockdown induced cellular senescence while reversed by p53 inactivation. ......................................................................................................... 37 Figure 5. The up-regulation of upstream stress response genes in LCTS1 knockdown WI-38 cells .......................................................................................................... 39 Figure 6. The DNA damage-induced p53 phosphorylation was increased in LCTS1 knockdown WI-38 cells ....................................................................................... 40 Figure 7. The cellular senescence after LCTS1 knockdown was not abrogated by P21 inhibition. ............................................................................................................. 42 Figure 8. The phosphorylation level of RB at Serine780 was decreased after LCTS1 knockdown. ......................................................................................................... 43 Maps and Table Contents 1. PLKO.1 shRNA expressing lentivirus vector..........................................................44 2. The oncogenic K-RAS cDNA was derived from pBabe-puro-K-RAS12V MMLV plasmid ................................................................................................................45 3. Oncogenic K-RAS expressing lentivirus vector.......................................................46 4. Real-time PCR primers............................................................................................47 5. Antibodies................................................................................................................47 6. shRNA clones from RNAi Consortium...................................................................48 | |
| dc.language.iso | en | |
| dc.title | 人類腫瘤抑制基因在肺細胞老化之研究 | zh_TW |
| dc.title | The role of a potential tumor suppressor in premature cellular senescence in a primary lung fibroblast cell line, WI-38 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林敬哲,陳美齡 | |
| dc.subject.keyword | 肺癌,細胞老化,p53, | zh_TW |
| dc.subject.keyword | lung cancer,cellular senescence,p53, | en |
| dc.relation.page | 52 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2014-08-19 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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