Please use this identifier to cite or link to this item:
標題: 探討p54nrb基因在非小細胞肺癌之上游調控區域
Characterization of p54nrb/NonO promoter in NSCLC
作者: 陳建宏
Chen, Chien-Hung
關鍵字: lung cancer;肺癌
出版社: 生物醫學研究所
引用: Babu, M. M., N. M. Luscombe, et al. (2004). "Structure and evolution of transcriptional regulatory networks." Curr Opin Struct Biol 14(3): 283-291. Barboro, P., A. Rubagotti, et al. (2008). "Differential proteomic analysis of nuclear matrix in muscle-invasive bladder cancer: potential to improve diagnosis and prognosis." Cell Oncol 30(1): 13-26. Basu, A., B. Dong, et al. (1997). "The intracisternal A-particle proximal enhancer-binding protein activates transcription and is identical to the RNA- and DNA-binding protein p54nrb/NonO." Mol Cell Biol 17(2): 677-686. Bohmann, D. (1990). "Transcription factor phosphorylation: a link between signal transduction and the regulation of gene expression." Cancer Cells 2(11): 337-344. Bucher, P. (1990). "Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences." J Mol Biol 212(4): 563-578. Chu, Y. W., P. C. Yang, et al. (1997). "Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line." Am J Respir Cell Mol Biol 17(3): 353-360. Clark, J., Y. J. Lu, et al. (1997). "Fusion of splicing factor genes PSF and NonO (p54nrb) to the TFE3 gene in papillary renal cell carcinoma." Oncogene 15(18): 2233-2239. Crick, F. (1970). "Central dogma of molecular biology." Nature 227(5258): 561-563. Dvir, A. (2002). "Promoter escape by RNA polymerase II." Biochim Biophys Acta 1577(2): 208-223. Emili, A., M. Shales, et al. (2002). "Splicing and transcription-associated proteins PSF and p54nrb/nonO bind to the RNA polymerase II CTD." RNA 8(9): 1102-1111. Fox, A. H., C. S. Bond, et al. (2005). "P54nrb forms a heterodimer with PSP1 that localizes to paraspeckles in an RNA-dependent manner." Mol Biol Cell 16(11): 5304-5315. Fry, C. J. and P. J. Farnham (1999). "Context-dependent transcriptional regulation." J Biol Chem 274(42): 29583-29586. Glantz, S. A. and W. W. Parmley (1992). "Passive smoking causes heart disease and lung cancer." J Clin Epidemiol 45(8): 815-819. Goldman, S. R., R. H. Ebright, et al. (2009). "Direct detection of abortive RNA transcripts in vivo." Science 324(5929): 927-928. Hallier, M., A. Tavitian, et al. (1996). "The transcription factor Spi-1/PU.1 binds RNA and interferes with the RNA-binding protein p54nrb." J Biol Chem 271(19): 11177-11181. Heinemeyer, T., X. Chen, et al. (1999). "Expanding the TRANSFAC database towards an expert system of regulatory molecular mechanisms." Nucleic Acids Res 27(1): 318-322. Iguchi, A., I. Kitajima, et al. (2000). "PEA3 and AP-1 are required for constitutive IL-8 gene expression in hepatoma cells." Biochem Biophys Res Commun 279(1): 166-171. Ishiguro, H., H. Uemura, et al. (2003). "55 kDa nuclear matrix protein (nmt55) mRNA is expressed in human prostate cancer tissue and is associated with the androgen receptor." Int J Cancer 105(1): 26-32. Jemal, A., A. Thomas, et al. (2002). "Cancer statistics, 2002." CA Cancer J Clin 52(1): 23-47. Khachigian, L. M., A. J. Williams, et al. (1995). "Interplay of Sp1 and Egr-1 in the proximal platelet-derived growth factor A-chain promoter in cultured vascular endothelial cells." J Biol Chem 270(46): 27679-27686. Kim, J. and D. J. Shapiro (1996). "In simple synthetic promoters YY1-induced DNA bending is important in transcription activation and repression." Nucleic Acids Res 24(21): 4341-4348. Kulkarni, S. J., A. F. Steinlauf, et al. (1988). "The dissonance mutant of courtship song in Drosophila melanogaster: isolation, behavior and cytogenetics." Genetics 118(2): 267-285. Kulozik, A. E., A. Bellan-Koch, et al. (1991). "Thalassemia intermedia: moderate reduction of beta globin gene transcriptional activity by a novel mutation of the proximal CACCC promoter element." Blood 77(9): 2054-2058. Kwok Fung Lo, A., Y. Liu, et al. (2001). "Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization." Biochim Biophys Acta 1520(2): 131-140. Le, C. H., Y. C. Ko, et al. (2001). "The heterogeneity in risk factors of lung cancer and the difference of histologic distribution between genders in Taiwan." Cancer Causes Control 12(4): 289-300. Lee, C. H., Y. C. Ko, et al. (2000). "Lifetime environmental exposure to tobacco smoke and primary lung cancer of non-smoking Taiwanese women." Int J Epidemiol 29(2): 224-231. Li, S., W. W. Kuhne, et al. (2009). "Involvement of p54(nrb), a PSF partner protein, in DNA double-strand break repair and radioresistance." Nucleic Acids Res 37(20): 6746-6753. Liang, S. and C. S. Lutz (2006). "p54nrb is a component of the snRNP-free U1A (SF-A) complex that promotes pre-mRNA cleavage during polyadenylation." RNA 12(1): 111-121. Mantovani, R. (1998). "A survey of 178 NF-Y binding CCAAT boxes." Nucleic Acids Res 26(5): 1135-1143. Matz, M., D. Shagin, et al. (1999). "Amplification of cDNA ends based on template-switching effect and step-out PCR." Nucleic Acids Res 27(6): 1558-1560. Miralles, F., L. G. Ofverstedt, et al. (2000). "Electron tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers in the nucleoplasm." J Cell Biol 148(2): 271-282. Ouhammouch, M., R. E. Dewhurst, et al. (2003). "Activation of archaeal transcription by recruitment of the TATA-binding protein." Proc Natl Acad Sci U S A 100(9): 5097-5102. Pavao, M., Y. H. Huang, et al. (2001). "Immunodetection of nmt55/p54nrb isoforms in human breast cancer." BMC Cancer 1: 15. Perkins, N. D., N. L. Edwards, et al. (1993). "A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation." EMBO J 12(9): 3551-3558. Prestridge, D. S. (1996). "SIGNAL SCAN 4.0: additional databases and sequence formats." Comput Appl Biosci 12(2): 157-160. Schug, J. (2008). "Using TESS to predict transcription factor binding sites in DNA sequence." Curr Protoc Bioinformatics Chapter 2: Unit 2 6. Sewer, M. B., V. Q. Nguyen, et al. (2002). "Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription." Endocrinology 143(4): 1280-1290. Sewer, M. B. and M. R. Waterman (2002). "Transcriptional complexes at the CYP17 CRS." Endocr Res 28(4): 551-558. Shav-Tal, Y. and D. Zipori (2002). "PSF and p54(nrb)/NonO--multi-functional nuclear proteins." FEBS Lett 531(2): 109-114. Straub, T., P. Grue, et al. (1998). "The RNA-splicing factor PSF/p54 controls DNA-topoisomerase I activity by a direct interaction." J Biol Chem 273(41): 26261-26264. Suzuki, Y., K. Yoshitomo-Nakagawa, et al. (1997). "Construction and characterization of a full length-enriched and a 5''-end-enriched cDNA library." Gene 200(1-2): 149-156. van Nimwegen, E. (2003). "Scaling laws in the functional content of genomes." Trends Genet 19(9): 479-484. Wang, Y. N. and W. C. Chang (2003). "Induction of disease-associated keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun." J Biol Chem 278(46): 45848-45857. Warnmark, A., E. Treuter, et al. (2003). "Activation functions 1 and 2 of nuclear receptors: molecular strategies for transcriptional activation." Mol Endocrinol 17(10): 1901-1909. Wen Cheng, Y. and H. Lee (2003). "Environmental exposure and lung cancer among nonsmokers: an example of Taiwanese female lung cancer." J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 21(1): 1-28. Whiteside, S. T. and S. Goodbourn (1993). "Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localisation." J Cell Sci 104 ( Pt 4): 949-955. Yang, Y. S., J. H. Hanke, et al. (1993). "NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss." Mol Cell Biol 13(9): 5593-5603. Zhang, Z. and G. G. Carmichael (2001). "The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs." Cell 106(4): 465-475. Zhu, B. Q., C. Heeschen, et al. (2003). "Second hand smoke stimulates tumor angiogenesis and growth." Cancer Cell 4(3): 191-196.
p54nrb/NonO is a multiple function nuclear protein. It has been implicated in numerous processes within the nucleus including transcriptional regulation, DNA unwinding, nuclear RNA processing, DNA double-strand break repair and tumorigenesis. In vivo, p54nrb usually forms a heterodimer with its partner PSF as a RNA splicing factor, but its roles in tumor progress is unknown. In recent years, p54nrb has been report in bladder cancer and prostate cancer that different expression of p54nrb between normal tissue and tumor tissue. p54nrb is upregulated in bladder cancer and prostate cancer. However, the mechanism about transcriptional regulation of p54nrb is still unknown. In this study, we characterize p54nrb function in non-small cell lung cancer cell (NSCLC) line and the transcriptional regulation of p54nrb promoter. First we find p54nrb transcription start site and use PCR technology to clone -1671/+1261 ( 2952 bp ) p54nrb promoter. Then we analyzed p54nrb promoter region by deletion of p54nrb promoter 5' end and luciferase report assay. The results of luciferase assay demonstrated that there is an important regulation region in p54nrb promoter +311/+414 fragment. And deletion of p54nrb promoter +109/+203 region can cause a part of luciferase activity decreasing. Furthermore, we used programe to predict what kind of transcription factor binding site on these two p54nrb promoter region. And we found three CCAAT box and AP-1, AP2α, Sp1, PEA3, NF-1, YY1 binding site. To investigate which transcription factors involve in transcriptional regulation of p54nrb. We observed the change of p54nrb after overexpressed AP-1, AP2α and Sp1 in CL1-0, CL1-5 and A549 cell line. But , the protein level and mRNA level of p54nrb don't change after overexpressed AP-1, AP2α and Sp1. On the other hand, our previous data showed that enforced p54nrb expression can suppress CL1-5 migration and invasion. In this study, we silenced p54nrb with siRNA and investigated the effect of migration and invasion. The result showed that there is no effect on migration and invasion after p54nrb silence. And so far, we still try to establish p54nrb silence stable clone.

p54nrb(Non-POU domain containing, octamer-bindin)在前人研究即被指出是一個多功能的核蛋白,參與多種細胞調節作用。其中包含轉錄調節、DNA的解旋、RNA修飾機制、雙股DNA修復與腫瘤新生。細胞內的p54nrb會與PSF形成複合體,並參與 pre-mature RNA的剪接作用,但p54nrb在癌症內所扮演的角色目前還不是很清楚。近幾年有研究指出,其中有研究指出在膀胱癌與前列腺癌的腫瘤組織與正常組織的p54nrb蛋白質表現量有明顯差異,腫瘤組織的p54nrb表現量會高於正常組織。然而目前對於p54nrb基因表現調控的機制還不是很清楚。本篇研究主要目的為定義出p54nrb基因在非小細胞肺癌之上游調控轉錄的區域,並分析可能的轉錄因子參與調控。首先我們找出p54nrb的轉錄起始位置,並利用聚合酶連鎖反應合成p54nrb啟動子-1671/+1261區域。接著利用5’缺失構築與冷光報導基因分析,結果發現p54nrb啟動子+311/+414區域為一段相當重要調控轉錄活性的區域,此外p54nrb啟動子+109/+203區域缺失也會造成部分啟動子的活性下降。進一步利用程式分析這兩個p54nrb啟動子片段,發現此區域具有三個CCAAT box與AP-1、AP2α、Sp1、PEA3、NF-1、YY1等轉錄因子結合位。為了進一步了解哪個轉錄因子可能參與到p54nrb基因的轉錄調控,我們將AP-1、AP2α、Sp1轉錄因子大量表現在CL1-0、CL1-5、A549肺癌細胞株,並觀察其對p54nrb基因表現的調控。根據實驗結果發現p54nrb的蛋白質與mRNA的表現量,在大量表現AP-1、AP2α、Sp1細胞中沒有明顯的差異。另外在實驗室先前的研究中發現大量表現p54nrb於CL1-5細胞株,會抑制其移動能力與侵襲能力。在本篇研究中,我們以siRNA將CL1-0、CL1-5細胞中的p54nrb靜默並觀察對細胞移動能力與侵襲能力的影響,結果發現在靜默p54nrb後對CL1-0、CL1-5細胞的移動能力與侵襲能力沒有顯著的影響。目前我們仍致力於挑選穩定靜默p54nrb的細胞株。
其他識別: U0005-2601201100175900
Appears in Collections:生物醫學研究所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.