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系統識別號 U0026-0812200912061134
論文名稱(中文) 細胞質磷脂水解酵素A2α型基因在IL-1β 刺激肺癌上皮細胞(A549)的後轉錄調控機制之研究
論文名稱(英文) Post-transcriptional regulation of cPLA2α mRNA expression under IL-1β treatment in A549 cells
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 94
學期 2
出版年 95
研究生(中文) 廖婉玲
研究生(英文) Wan-Lin Liao
電子信箱 ashley0214999@yahoo.com.tw
學號 s2693406
學位類別 碩士
語文別 中文
論文頁數 88頁
口試委員 口試委員-呂增宏
指導教授-張文昌
指導教授-曾大千
中文關鍵字 後轉錄基因調控 
英文關鍵字 post-transcriptional regulation 
學科別分類
中文摘要 cPLA2α(cytosolic phospholipase A2 α)是位於細胞內的一種磷質水解酵素,是調節arachidonic acid釋放的限制速率酵素。本實驗室在先前的實驗中發現,在人類肺癌細胞(A549)中處理IL-1β可以增加cPLA2α mRNA及蛋白質的表現量,分別有5倍及2倍的增加。但在reporter assay的實驗中則看到cPLA2 α啟動區的活性僅有1.4倍的增加。因此我們便想探討在IL-1β的刺激下,cPLA2 α mRNA及protein的表現量增加,是否是透過後轉錄基因調控(post-transcriptional regulation)的方式來增加cPLA2α mRNA的穩定度,進而促使其表現量的增加。

過去,對於基因的表現都認為它是在DNA或蛋白質的層級上作調控,但是近年來,越來越多的人認為基因的調控也可以發生在RNA的層級上,因此後轉錄基因調控的概念漸漸被提出,後轉錄基因調控牽涉兩個層面:一個是和轉譯的效率(translational efficiency)有關,另一個則是探討mRNA的穩定度(mRNA stability)。

在一些受到刺激而會快速表現的基因中,可以發現在其mRNA的3’端非轉譯區(3’-UTR)具有AU-rich element的序列。且此AU-rich element的序列也被報導和該基因短暫的半生期有關。比較人類、大鼠和小鼠 cPLA2α mRNA 3’端非轉譯區的序列可以發現在這些物種中都帶有一致的AU-rich motif,顯示其在調控RNA的穩定上扮演一個重要的角色。文獻報導也指出當某一些RNA結合蛋白結合到上述的AU-rich element時,則可扮演穩定其 mRNA的角色,而其中HuR是目前研究顯示調控RNA 穩定的重要蛋白。HuR 為ELAV-like family中的一員,具有三個RNA recognition motif(RRM)及HuR nuclear shuttling domain (HNS)。因此有報導指出它除了結合並穩定mRNA之外,也具有將跟其結合的mRNA從細胞核帶到細胞質中的功能。

在本論文中,經由in vitro RNA-EMSA的實驗我們已確認cPLA2α的3’端非轉譯區中與RNA結合蛋白的結合區域,並證明與此區域結合的蛋白質為HuR。再利用in vitro蛋白表現的方式,發現GST-HuR的蛋白會結合到cPLA2 α的3’端非轉譯區。利用RNA免疫沈降法(RNA-immunoprecipitation)的實驗也可看到HuR結合到cPLA2α的現象會隨著IL-1β的刺激而增加。利用siRNA將HuR knockdown,發現原本受到IL-1β誘導而增加的cPLA2α mRNA的表現則無法再受到誘導。綜合以上結果,我們推論IL-1β對cPLA2α的調控除了略微提高其轉錄效能外,更重要的是透過增加其mRNA的穩定度,而HuR在這穩定的機制上也扮演一個重要的調控角色。



英文摘要 cPLA2 α (cytosolic phospholipase A2 α) is the major intracellular form of PLA2, which selectively hydrolyzes membrane phospholipids at the sn-2 position, and is the rate-limiting enzyme in the eicosanoid production. In our previous study, we found that IL-1β, a pro-inflammatory cytokine, can increase the expression of cPLA2α mRNA and protein in A549 cells. However, we found that the promoter activity of cPLA2α gene cannot be induced by IL-1β significantly. We were interested in understanding the mechanism regulating the expression level of cPLA2 α.

In addition to transcriptional regulation, the post-transcriptional regulation of mRNA turnover has emerged as a key step in the regulation of eukaryotic gene expression. Although the mechanisms determining mRNA turnover are not well established, they are generally believed to involve RNA-binding proteins (trans-acting factors) recognizing specific RNA sequences (cis-elements). By cloning the 3’-UTR of cPLA2 α into the reporter vector, we found that the reporter activity was down-regulated. These results indicated that this region contains cis-elements that are known to regulate the mRNA stability. From the RNA-EMSA assay, we located the cis-element at the 3’-UTR region from +2558 bp to +2791 bp and this region contains a lot of AU-rich elements (ARE). Furthermore, we identified that HuR, an mRNA stabilizing protein known to bind the ARE, can bind to this region as demonstrated by the antibody-shift assay. The same result was also obtained from in vitro binding assays when we used the GST-HuR fusion protein to perform the experiments. In addition, we observed that IL-1β enhanced the endogenous binding between HuR and cPLA2α mRNA as seen from the RNA-immunoprecipitation assay. Based on these results, we conclude the notion that HuR plays an important regulatory role in stabilizing cPLA2α mRNA especially when A549 cells were treated with IL-1β.








論文目次 考試合格證明…………………………………………………………………... I

中文摘要………………………………………………………………………... II

英文摘要…………………………………………………………....................... IV

圖目錄………………………………………………………………………....... VII

附錄……………………………………………………………………………… VII

縮寫指引………………………………………………………………………… IX



第一章 緒論…………………………………………………………………... 1
第二章 實驗材料及方法
來源…………………………………………………………………... 11
實驗方法……………………………………………………………... 16
第三章 實驗結果
第一節 IL-1β對於誘導cPLA2α啟動區的活性並不顯著................. 34
第二節 探討有無trans-acting factors結合至cPLA2α mRNA
3’端非轉譯區域(3’-UTR)....................................................
35
第三節 確認結合在cPLA2α mRNA 3’端非轉譯區的trans-acting factors為何..............................................................................
35
第四節 確認結合在cPLA2α mRNA 3’端非轉譯區(3’-UTR)的結合蛋白為HuR……………………………………………….
36
第五節 IL-1β可促進HuR結合至cPLA2α的mRNA……………… 37
第六節 HuR knockdown 對cPLA2α mRNA的影響………………. 37
第七節 IL-1β可誘導HuR選擇性的結合到與發炎反應有關
的基因的mRNA………………………………………….....
38


第四章 討論…………………………………………………………...……… 40
第五章 參考文獻……………………………………………………...……… 44

附圖
Fig. 1. Effect of IL-1β on the promoter activity of cPLA2α gene…………… 53
Fig. 2. Identification of the binding region in the 3’-UTR of
cPLA2α mRNA…………………………………………………………
54
Fig. 3. Characterization of the trans-acting factors bound with
cPLA2α mRNA…………………………………………………………
55
Fig. 4. The expression level of HuR in A549 cells…………………………... 56
Fig. 5. HuR bound to the 3’-UTR of cPLA2α mRNA in vitro……………….. 57
Fig. 6. HuR bound to the 3’-UTR of cPLA2α mRNA in vivo……………….. 58
Fig. 7. Down-regulation of HuR affected the expression of cPLA2α
under IL-1β treatment………………………………………………….
59
Fig. 8. HuR selectively bound to the mRNA involved in inflammation…….. 60


附錄
附錄一. Typical steps in the control of gene expression..................................... 61
附錄二. Regulation of transferrin receptor mRNA stability by IRE/IRP interactions in the 3’-UTR……………………………………………. 62
附錄三. AU-rich sequence has the destabilizing effect on mRNA half-life…… 63
附錄四 Classification of ARE sites…………………………………………… 64
附錄五. ARE-binding protein………………………………………………….. 65
附錄六. Model for how the A+U-rich mediates stability and instability………. 66
附錄七. Mammalian PLA2 enzymes………………………………………... 67
附錄八. Structure of cPLA2α isoform………………………………………. 68
附錄九. A schematic model for cPLA2α activation in cells………………… 69
附錄十. Synthesis and structure of eicosanoids…………………………….. 70
附錄十一. Alignment of ARE-containing sequence in the 3’-UTR of the
cPLA2 gene in rat, mouse and human species……………………… 71
附錄十二. The designed primers in the 3’-UTR of human cPLA2α gene……… 72
附錄十三. The promoter and multiple cloning sequence of the
pGEM-T Easy Vectors………………………………………………
73
附錄十四. Time-depend effect of IL-1β on the expression of cPLA2α
mRNA in A549 cells……………………………………………….. 74
附錄十五. Time-dependent effect of IL-1β on the expression of cPLA2α
protein in A549 cells………………………………………………..
75
附錄十六. Effect of IL-1β on cPLA2α mRNA stability………………………... 76
附錄十七. Structure of reporter gene constructs with cPLA2α 3’-UTR……….. 77
附錄十八. Inclusion of sequence from the 3’-UTR of cPLA2α in the chimeric reporter message causes a decrease in luciferase activity………….
78
附錄十九. The 3’-UTR of cPLA2α confer IL-1β inducibility to
reporter constructs………………………………………………….
79


參考文獻 Brennan, C. M., and Steitz, J.A. HuR and mRNA stability. Cell Mol. Life Sci. 58: 266-277, 2001.

Bonventre, J.V. The 85-KD cytosolic phospholipase A2 knockout mouse: a new tool for physiology and cell biology. J. Am. Soc. Nephrol. 10: 404-412, 1999.

Bonventre, J.V. Phospholipase A2 and signal transduction. J. Am. Soc. Nephrol. 3: 128-150, 1992.

Chen, C.Y., Del, Gatto-Konczak F., Wu, Z., and Karin, M. Stabilization of interleukin-2 mRNA by the c-jun NH2-terminal kinase pathway. Science 280: 1945-1949, 1998

Chen, C.Y., and Shyu, A. B. Au-rich elements:characterization and importance in mRNA degradation. Trends Biochem. Sci. 20: 465-470, 1995.

Croxtall, J.D., Newman, S.P., Choudhury, Q., and Flower, R.J. The concerted regulation of cPLA2, COX2, and lipocortin 1 expression by IL-1β in A549 cells. Biochem. Biophys. Res. Comm. 220: 491-495, 1996.

Chung, S., Jiang, L., Cheng, S., and Furneaux, H. Purification and Properties of HuD, a Neuronal RNA-binding Protein. J. Biol. Chem. 271: 11518-11524, 1996.

Chepenid K.P., Diaz, A., and Jimenez, S.A. Epidermal growth factor coordinately regulates the expression of prostaglandin G/H synthase and cytosolic phospholipase A2 genes in embryonic mouse cell. J. Biol. Chem. 269: 21786-21792, 1994.

Carol, J. Wilusz, M. W., and Stuart, W. P. The cap-to-tail guide to mRNA turnover. Nature Rev. Mol. Cell Biol. 2: 237-246, 2001.

Dixon, D.A., Tolley, N.D., King, P.H., Nabors, L.B., McIntyre, T.M., Zimmerman, G.A., and Prescott, S.M. Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells. J. Clin. Invest.108: 1657-1665, 2001.

Dolan-O’Keefe, M., and Nick, H.S. Inhibition of cytoplasmic phospholipase A2 expression by glucocortocoid in rat intestinal epithelial cells. Gastroenterology 6: 855-864, 1999.

Dennis, E.A. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22: 1-2, 1997.

Fouda, S.I., Molski, T.F.P., Ashour, M.S., and Sha fi, R.I. Effect of lipopolysaccharide on mitogen-activated protein kinases and cytosolic phospholipase A2. Biochem. J. 308: 815-822, 1995.

Hentze, M. W., and Kuhn, L.C. Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide and oxidative stress. Proc. Natl. Acad. Sci. USA 93: 8175-8182, 1996.

Helene, T., Karim, C., and Jamal, T. mRNA degradation machines in eukaryotic cells. Biochimie 84: 821-837, 2002.

Hirabayashi, T., and Shimizu, T. Localization and regulation of Cytosolic phospholipase A2. Biochim. Biophys. Acta 1488: 124, 2000.

Hoeck, W.G., Ramesha, C.S., Chang, D.J., Fan, N., and Heller, R.A. Cytoplasmic phospholipase A2 activity and gene expression are stimulated by tumor necrosis factor: dexamethasone blocks the induced synthesis. Proc. Natl. Acad. Sci. USA 90: 4475, 1993.

Hefner, Y., Borsch-Haubold, A.G., Murakami, M., Wilde, J.I., Pasquet, S., Schieltz, D., Ghomashchi, F., Yates III, J. R., Armstrong, C. G., Paterson, A., Cohen, P., Fukunaga, R., Hunter, T., Kudo, I., Watson, S. P., and Gelb, M. H. Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases. J. Biol. Chem. 275: 37542–37551, 2000.

Hulkower, K.I., Wertheimer, S.J., Levin, W., Coffey, J.W., Anderson, C.M., Chen, T., Dewitt, D.L., Crowl, R.M., Hope, W.C., and Morgan, D.W. Interleukin-1 induces cytosolic phospholipase A2 and prostaglandin H synthase in rheumatoid synnovial fibroblasts. Arthritis Rheum 37: 653-661, 1994.

Hernandez, M., Bayon, Y., Sanchez, Crespo M., and Nieto, M.L. Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1. Biochem. J. 328: 263-269, 1997.

Hitti, E., Iakovleva, T., Brook, M., Deppenmeier, S., Gruber, A.D., Radzioch, D., Clark, A.R., Blackshear, P.J., Kotlyarov, A., and Gaestel, M. Miotogen-activtated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mRNA stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine-rich element. Mol. Cell. Biol. 26: 2399-2407, 2006.

Jacobson, A., and Peltz, S. W. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Ann. Rev. Biochem. 65: 693-739, 1996.

Jonathon, L. E. Dean, Robin, W., Kamal, R. M., Gareth. S., Andrew R. C., and Jeremy, S. The 3’ untranslated region of tumor necrosis factor alpha mRNA is a target of the mRNA-stabilizing factor HuR. Mol. Cell. Biol. 21: 721-730, 2000.

Jack, D. keene and Patrick, J. Lager. Post-transcriptional operons and regulons co-ordinating gene expression. Chromosome Res. 13: 327-337, 2005.

Juan, M., Samuel M., and Ju¨rg Ba¨hler. Post-transcriptional control of gene expression: a genome-wide perspective. Trends Biochem. Sci. 30: 506-514, 2005.

Kudo, I., and Murakami, Ma. Phospholipase A2 enzymes. Prostaglandins other Lipid Mediat. 68-69: 3-58, 2002.

Gantt, K., Cherry, J., Tenny, R., karschner, V., and Pekala, P.H. An early event in adipogenesis, the nuclear selection of the CCAAT enhancer-binding protein β(C/EBPβ) mRNA by HuR and its translocation to the cytosol. J. Biol. Chem. 280: 24768-24774, 2005.

Loflin, P.T., Chen, C.Y., Xu, N., and Shyu, A. B. Transcriptional pulsing approaches for analysis of mRNA turnover in mammalian cells. Methods 17: 11-20, 1999.

Lin, L.L., Lin, A.Y., and DeWitt, D.L. Interleukin-1α induces the accumulation of cytosolic phospholipase A2 and the release of prostaglandin E2 in human fibroblasts. J. Biol. Chem. 267: 23451, 1992.

Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269–278, 1993.

Levy, N.S., Chung, S., Furneaux, H., and Levy, A.P. Hypoxi Stabilization of Vascular Endothelial Growth Factor mRNA by the RNA-binding Protein HuR. J. Biol. Chem.273: 6417-6423, 1998.

Lesile, C.C. Properties and regulation of cytosolic phospholipase A2. J. Biol. Chem. 272: 16709-16712, 1997.

Ma, W. J., Chung, S., and Furneaux, H. The Elav-like proteins bind to AU-rich elements and to the poly(A) tail of mRNA. Nucl. Acids Res. 25: 3564-3569, 1997.

Myer, V.E., Fan, X.C., and Steitz, J.A. Identification of HuR as a protein implicated in AUUUA-mediated mRNA decay. EMBO J. 16: 2130-2139, 1997.

Maxwell, A.P., Goldberg, H.J., Tay, A.H., Li, Z.G., Arbus, G.S., and Skorecki, K.L. Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells. Biochem. J. 295: 763, 1993.

Miyashita, A., Crystal, R.G., and Hay, J.G. Identification of a 27 bp 5,- flanking region element responsible for the low level constitutive expression of the human cytosolic phospholipase A2 gene. Nucl. Acids Res. 23: 293, 1995.

Muthalif, M. M., Hefner, Y., Canaan, S., Harper, J., Zho, H., Parmentier, J. H., Aebersold, R., Gelb, M. H., and Malik, K. U. Functional interaction of calcium/calmodukin-dependent protein kinase II and cytosolic phospholipase A2. J. Biol. Chem. 276: 39653–39660, 2001.

Nakamura, T., Lin, L.L., Kharbanda, S., Knopf, J., and Kufe, D. Macrophage colony stimulating factor activates phosphatidylcholine hydrolysis by cytoplasmic phospholipase A2. EMBO J. 11: 4917, 1992.
Nakamura, T., Lin, L.L., Kharbanda, S., Knopf, J., and Kufe, D.
Macrophage colony stimulating factor activates phosphatidylcholine hydrolysis by cytoplasmic phospholipae A2. EMBO J. 11: 4917-4922, 1992.

Peltz, S.W., Brewer, G., Bernstein, P., Hart, P. A., and Ross, J. Regulation of mRNA turnover in eukaryotic cells. Crit. Rev. Eukaryot. Gene Expr. 1: 99-126, 1991

Peng, S.S., Cheng, C.Y., Xu, N., and Shyu, A.B. RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein. EMBO J. 17: 3461-3470, 1998.

Park, S., Myszka, D.G., Yu, M., Littler, S.J., Laird-Offringa IA. HuD RNA recognition motifs play distinct roles in the formation of a stable complex with AU-rich RNA. Mol. Cell. Biol. 20: 4765-4772, 2000.

Qiu, Z. H., Gijon, M.A., de Carvalho, M.S., Spender, D.M., and Leslie, C.C. The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acids release in macrophage. J. Biol. Chem. 273: 8203-8211, 1998.

Ross, J. Control of messenger RNA stability in higher eukaryotes. Trends Genet. 12: 171–175, 1996.

Standart, N. J., and Jackson, R. J. Regulation of translation by specific protein/mRNA interaction. Biochemie 76: 867-879, 1994.

Sachs, A.B. Messenger RNA degradation in eukaryotes. Cell 74: 413-421, 1993.

Shyu, A. B., Greenberg, M. E., and Belasco, J. G. The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. Genes Dev. 3: 60-72, 1989.

Schalkwijk, C., Vervoordeldonk, M., Pfeilschifter, J. and van den Bosch, H. Interleukin-1β-induced cytosolic phospholipase A2 activity and protein synthesis is blocked by dexamethasone in rat mesangial cells. FEBS Lett. 333: 339-343, 1993.

Stacy, A. B., Marilee, W., Christina, D., and Raphael, A.N. Induction of cPLA2 in lung epithelial cells and non-small cell lung cancer is mediated by Sp1 and c-Jun. J. Biol. Chem. 276: 42737-42743, 2001.

Sengupta, S., Jang, B.C., Wu, M.T., Paik, J.H., Furneanx, H., and Hla, T. The RNA-binding protein HuR regulates the expression of cyclooxygenase 2. J. Biol. Chem. 278: 25227-25233.

Tay, A., Maxwell ,P., Li, Z., Goldberg, H., and Skorecki, K. Isolation of promoter for cytosolic phospholipase A2 (cPLA2). Biochim. Biophys. Acta 1217: 345, 1994.

Tay, A., Maxwell, P., Li, Z., Goldberg, H., and Skorecki, K. Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated posttranscriptionally. Biochem. J. 304: 417-422, 1994.

Wilson, T., and Treisman, R. Removal of poly(A) and and consequent degradation of c-fos mRNA facilitated by 3’ AU-rich sequences. Nature 336: 396-399, 1988.

Wu, T., Levine, S.J., Lawrence, M.G., Logun, C., Angus, C.W., and Shelhamer, J.H. Interferon-γ induces the synthesis and activation of cytosolic phospholipase A2. J. Clin. Invest. 93: 571, 1994.

Wu, T., Ikezono, T., Angus, C.W., and Shelhamer. J.H. Tumor necrosis factor-α induces the 85-kDa cytosolic phospholipase A2 gene expression in human bronchial epithelial cells. Biochim. Biophys. Acta 1310: 175, 1996.

Wu, T., Ikezono, T., Angus, C.W., and Shelhamer, J.H. Characterization of the promoter for the human 85-kDa cytosolic phospholipase A2 gene. Nucl. Acids Res. 22: 5093, 1994.

Wang, W., Caldwell, M.C., Lin, S., Furneaux, H., and Gorospe, M., HuR regulates cyclinA and cyclin B1 mRNA stability during cell proliferation. EMBO J. 19: 2340-2350, 2000.

Zhao, Z. Chang, F.C., Furneaux, H.M. The identification of an endonuclease that cleaves within an HuR binding site in mRNA. Nucl. Acids Res. 28: 2695-2701, 2000.

王薇喬 Transcriptional regulation of human cytosolic phospholipase A2α gene. 國立成功大學藥理學研究所碩士論文, 2004.






















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