人肝癌细胞中DHRS4LI选择性剪切新亚型的鉴定和分析
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摘要
全反式维甲酸(all-trans Retinoic Acid,atRA)是体内类激素样生理活性物质,在哺乳动物包括人类,成体内atRA浓度过高或过低都可能会导致各种疾病和肿瘤的发生。在体内,atRA是由维生素A(vitamin A )也称视黄醇(Retinol)经两步代谢生成,其中间代谢产物为视黄醛(Retinal)。成体内Retinal的生理作用剂量范围也很窄,过高或过低均可会对机体产生不利影响。因此,体内atRA和Retinal的代谢均受到严格精细的调控作用,整个代谢过程是一个由多酶参与的调节过程。
近年来本课题组一直致力于一种全称为辅酶Ⅱ依赖性视黄醇脱氢/还原酶(NADP(H)- dependent retinol dehydrogenase/ reductase,NRDR)的短链脱氢酶(short-chain dehydrogenase /reductase,SDR)的研究。NRDR最早从兔肝细胞质溶胶中纯化得到,是atRA代谢中的限速酶,具有很高的视黄醇氧化、视黄醛还原活性,且广泛分布于哺乳动物组织中。较之于短链脱氢酶家族中已知的其它成员,NRDR在生理条件下表现出更强的视黄醛还原活性,因此,它在维持体内RA及Retinal的代谢平衡中发挥重要的调节作用。
Genebank中登陆号为AB045133的cDNA被认为是NRDR的编码基因,称为DHRS4。人DHRS4基因簇有三个基因拷贝,分别为DHRS4,DHRS4L2与DHRS4L1(又称为DHRS4X),其中前二者相似性很高(90%-98%;≥1kb)属于片段复制(Segmental Duplication)。DHRS4L1与DHRS4和DHRS4L2之间的同源性分别为77.8%和77.7%。在研究基因表达与调控的过程中,我们发现DHRS4基因簇的3个拷贝基因存在非常复杂的转录及选择性剪接方式,从而产生多种选择性剪接亚型,这些剪接亚型在不同组织中表达情况的差异具有重要的临床意义。本研究从人肝癌细胞HepG2中鉴定出DHRS4L1选择性剪接新亚型,检测其在不同组织和细胞中的表达情况,并探讨其临床意义。
我们首先采用RACE(cDNA末端快速扩增法)方法成功地从HepG2细胞中获得选择性剪接新亚型的3’末端cDNA序列,鉴定其结构推测其5’末端cDNA序列,进而得到其cDNA序列,全长1117bp;运用生物信息学方法对新亚型序列进行分析比对发现,该新亚型的cDNA序列与DHRS4LI基因高度同源,命名为DHRS4L1A1;通过人基因组Blast分析,该新亚型DHRS4L1A1由于选择性剪接造成了第3~7外显子丢失,并保留第2外显子和第3外显子之间一段长度为85bp内含子序列,结构为E1-E2-insertion-E8-E9-E10;通过查找开放阅读框架,发现该剪接新亚型的可阅读框架在1~699bp之间,预测其编码的蛋白质序列为232个氨基酸,具有SDR家族2个保守的模序TAXXXGXG与YXXSK及过氧化物酶体定位信号SRL。用RT-PCR方法对该剪接新亚型在8种常见的细胞株中的表达情况进行半定量分析,结果显示该新亚型在肿瘤细胞中的表达明显高于正常细胞,尤其在卵巢癌细胞SKOV3中的表达量显著高于其他肿瘤细胞,提示DHRS4L1A1可能参与生殖系统相关酶类的代谢。
All-trans retinoic acid (atRA)is a hormone-like and physiological active substance in mammals, including humans. In vivo, either too high or too low concentration of the atRA may lead to various disease and cancer. atRA is derived from vitamin A, which also known as retinol, through a two-step oxidative metabolism, and retinol as intermediate metabolite between the two metabolisms, has a narrow physiological dose range in vivo, both too high or too low will also exert negative effects on organism. Therefore, the metabolism of both atRA and retinol are subject to strict and subtle regulation.
In recent years, our group has been committed to the study on an short-chain dehydrogenase /reductas(eSDR)which is called NADP(H)-dependent retinol dehydrogenase/ reductase (NRDR). NRDR identified from rabbit liver cytosol firstly is a rate-limiting enzyme during metabolism of atRA. It has high retinol oxidation and retinal reduction activity, and is widely distributed in various mammalian tissues.
Compared with other members of SDR superfamily, NRDR exhibits higher aldehyde reductase activity in retinoic acid (RA) metabolism. Therefore , NRDR may paly an important role in keeping the metabolism balance between atRA and retinal in vivo.
We focused on the cDNA that deposited in Genebank (accession number:AB045133) and annotated as the coding gene of NRDR, which is called DHRS4. There are three copies for human DHRS4 gene cluster, including DHRS4, DHRS4L2 and DHRS4L1. The identity between the former two copies is 97.5%, suggesting that they are segmental duplication (90%-98%;≥1 kb). In the study on gene expression and regulation of DHRS4, we found there are sophisticated ways of transcription and alternative splicing, and obtained many alternatively spliced variants. The different expression of these spliced variants in different tissues may have important clinical significance. In this study, we identified an alternatively spliced variant from Hep-G2, detected its expression in different tissues and cells, as well as its clinical significance.
In this study, we cloned the full-length cDNA of this alternatively spliced variant in Hep-G2 cell line by Rapid amplification of cDNA ends(RACE), it’s about 1117bp. Sequence alignment analysis shows that the cDNA of this alternatively spliced variant named DHRS4L1A1 has highly homology with DHRS4L1. Human genome Blast analysis shows that the structure of DHRS4L1A1 is e1-e2-insertion-e8-e9-e10 with exon 3 to exon 7 deletion. We discovered an open reading frame of 699bp, and it may could be translated a protein contained 232 amino acids. Finally, we found DHRS4L1A1 could express in many different cell lines , and its expression in tumor cells ,specifically in ovarian cancer cell line SKOV3, is higher than normal cells, suggesting the enzyme might has close relationship with metabolic pathways of reproductive system.
近年来本课题组一直致力于一种全称为辅酶Ⅱ依赖性视黄醇脱氢/还原酶(NADP(H)- dependent retinol dehydrogenase/ reductase,NRDR)的短链脱氢酶(short-chain dehydrogenase /reductase,SDR)的研究。NRDR最早从兔肝细胞质溶胶中纯化得到,是atRA代谢中的限速酶,具有很高的视黄醇氧化、视黄醛还原活性,且广泛分布于哺乳动物组织中。较之于短链脱氢酶家族中已知的其它成员,NRDR在生理条件下表现出更强的视黄醛还原活性,因此,它在维持体内RA及Retinal的代谢平衡中发挥重要的调节作用。
Genebank中登陆号为AB045133的cDNA被认为是NRDR的编码基因,称为DHRS4。人DHRS4基因簇有三个基因拷贝,分别为DHRS4,DHRS4L2与DHRS4L1(又称为DHRS4X),其中前二者相似性很高(90%-98%;≥1kb)属于片段复制(Segmental Duplication)。DHRS4L1与DHRS4和DHRS4L2之间的同源性分别为77.8%和77.7%。在研究基因表达与调控的过程中,我们发现DHRS4基因簇的3个拷贝基因存在非常复杂的转录及选择性剪接方式,从而产生多种选择性剪接亚型,这些剪接亚型在不同组织中表达情况的差异具有重要的临床意义。本研究从人肝癌细胞HepG2中鉴定出DHRS4L1选择性剪接新亚型,检测其在不同组织和细胞中的表达情况,并探讨其临床意义。
我们首先采用RACE(cDNA末端快速扩增法)方法成功地从HepG2细胞中获得选择性剪接新亚型的3’末端cDNA序列,鉴定其结构推测其5’末端cDNA序列,进而得到其cDNA序列,全长1117bp;运用生物信息学方法对新亚型序列进行分析比对发现,该新亚型的cDNA序列与DHRS4LI基因高度同源,命名为DHRS4L1A1;通过人基因组Blast分析,该新亚型DHRS4L1A1由于选择性剪接造成了第3~7外显子丢失,并保留第2外显子和第3外显子之间一段长度为85bp内含子序列,结构为E1-E2-insertion-E8-E9-E10;通过查找开放阅读框架,发现该剪接新亚型的可阅读框架在1~699bp之间,预测其编码的蛋白质序列为232个氨基酸,具有SDR家族2个保守的模序TAXXXGXG与YXXSK及过氧化物酶体定位信号SRL。用RT-PCR方法对该剪接新亚型在8种常见的细胞株中的表达情况进行半定量分析,结果显示该新亚型在肿瘤细胞中的表达明显高于正常细胞,尤其在卵巢癌细胞SKOV3中的表达量显著高于其他肿瘤细胞,提示DHRS4L1A1可能参与生殖系统相关酶类的代谢。
All-trans retinoic acid (atRA)is a hormone-like and physiological active substance in mammals, including humans. In vivo, either too high or too low concentration of the atRA may lead to various disease and cancer. atRA is derived from vitamin A, which also known as retinol, through a two-step oxidative metabolism, and retinol as intermediate metabolite between the two metabolisms, has a narrow physiological dose range in vivo, both too high or too low will also exert negative effects on organism. Therefore, the metabolism of both atRA and retinol are subject to strict and subtle regulation.
In recent years, our group has been committed to the study on an short-chain dehydrogenase /reductas(eSDR)which is called NADP(H)-dependent retinol dehydrogenase/ reductase (NRDR). NRDR identified from rabbit liver cytosol firstly is a rate-limiting enzyme during metabolism of atRA. It has high retinol oxidation and retinal reduction activity, and is widely distributed in various mammalian tissues.
Compared with other members of SDR superfamily, NRDR exhibits higher aldehyde reductase activity in retinoic acid (RA) metabolism. Therefore , NRDR may paly an important role in keeping the metabolism balance between atRA and retinal in vivo.
We focused on the cDNA that deposited in Genebank (accession number:AB045133) and annotated as the coding gene of NRDR, which is called DHRS4. There are three copies for human DHRS4 gene cluster, including DHRS4, DHRS4L2 and DHRS4L1. The identity between the former two copies is 97.5%, suggesting that they are segmental duplication (90%-98%;≥1 kb). In the study on gene expression and regulation of DHRS4, we found there are sophisticated ways of transcription and alternative splicing, and obtained many alternatively spliced variants. The different expression of these spliced variants in different tissues may have important clinical significance. In this study, we identified an alternatively spliced variant from Hep-G2, detected its expression in different tissues and cells, as well as its clinical significance.
In this study, we cloned the full-length cDNA of this alternatively spliced variant in Hep-G2 cell line by Rapid amplification of cDNA ends(RACE), it’s about 1117bp. Sequence alignment analysis shows that the cDNA of this alternatively spliced variant named DHRS4L1A1 has highly homology with DHRS4L1. Human genome Blast analysis shows that the structure of DHRS4L1A1 is e1-e2-insertion-e8-e9-e10 with exon 3 to exon 7 deletion. We discovered an open reading frame of 699bp, and it may could be translated a protein contained 232 amino acids. Finally, we found DHRS4L1A1 could express in many different cell lines , and its expression in tumor cells ,specifically in ovarian cancer cell line SKOV3, is higher than normal cells, suggesting the enzyme might has close relationship with metabolic pathways of reproductive system.
引文
[1] Lutan, R. Retinoids in cancer chemoprevention. FASEB J. 1996,10: 1031-1039.
[2] Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J. 1996,10:940-954.
[3] Mangelsdorf DJ, Thummel C, Beato M. The nuclear receptor superfamily: the second decade. Cell 1995,83:835-839.
[4] Altucci L, Gronemeyer H. Nuclear receptors in cell life and death. Trends Endocrinol. Metab. 2001,12:460-468
[5] Boulogne B, Levacher C, Durand P. Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: Immnolocalization and implication in the control of the number of gonocytes. Biol. Reprod. 1999,61:1548-1557.
[6] Mendelsohn C, Lohnes D, Decimo D. Function of the retinoic acid receptors (RARs) during development(II).Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development. 1994,120:2749-2771.
[7] Malpel S, Mendelsohn C, Cardoso WV. Regulation of retinoic acid signaling during lung morphogenesis. Development 2000,127:3057-3067.
[8] Huang, M.E., Ye, Y-C., Chen, S-R., Chai, J-R.,Lu, J-X., Shoa, Lin, Guo, L-J., and Wang, Z-Y. (1988) Use of all-transretinoic acid in the treatment of acute promyelocytic leukemia.Blood 72, 567-572.
[9] Colucciello M. Pseudotumor cerebri induced by all-trans retinoic acid treatment of acute promyelocytic leukemia. Arch Ophthalmol. 2003,121: 1064-1069.
[10] Recchia F, Saggio G, Nuzzo A, Biondi E, Di-Blasio A, Cesta A, Candeloro G, Alesse E, Rea S. Multicenter phase 2 study of interleukin-2 and 13-cis retinoic acid as maintenance therapy in advanced non-small-cell lung cancer. J Immunother. 2006, 29: 87-94.
[11] Brtko J, Thalhamer J. Renaissance of the biologically active vitamin A derivatives: established and novel directed therapies for cancer and chemoprevention. Curr Pham Des 2003, 9: 2067-2077.
[12] Grubbs CJ, Hill DL, Bland KI, Beenken SW, Lin TH, Eto I, Atigadda VR, Vines KK, Brouillette WJ, Muccio DD. 9cUAB30, an RXR specific retinoid, and/or tamoxifen in the prevention of methylnitrosourea-induced mammary cancers. Cancer Lett. 2003,201: 17-24.
[13] Kannan K, Khan HA, Jain R, Hussein SS, Dennison D. All-trans retinoic acid-induced myositis. Br J Haematol 2005,131: 560-569.
[14] Zhong JC, Huang DY, Liu GF, Jin HY,Yang YM, Li YF, Song XH, Du K. Effect of all-trans retinoic acid on orphan receptor APJ signaling in spontaneously hypertensive rats. Cardiovascular Res 65(3): 743-750 (2005).
[15] JC Zhong, DY Huang, YM Yang, YF Li, GF Liu, XH Song, K Du. Upregulation of angiotensin-converting enzyme 2 by all-trans retinoic acid in spontaneously hypertensive rats. Hypertension 44:907-912 (2004).
[16] Yan-Mei Yang, Dong-Yang Huang, Ge-Fei Liu, Jiu-Chang Zhong, Yi-Fan Li, Xu-Hong Song. Inhibitory Effects of Vitamin A on TCDD-induced Cytochrome P-450 1A1 Enzyme Activity and Expression. Toxicological Sciences 85:727-734 (2005).
[17] Yan-Mei Yang, Dong-Yang Huang, Ge-Fei Liu, Jiu-Chang Zhong, Yi-Fan Li, Xu-Hong Song. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on vitamin A metabolism in mice. J Biochem Molecular Toxicology 19:327-335 (2005).
[18] Liang B, Song XH, Liu GF, Li R, Xie JP, Xiao LF, Du MD, Zhang QX,Xu XY, Gan XQ, Huang DY. Involvement of TR3/Nur77 translocation to the endoplasmic reticulum in ER stress-induced apoptosis. Experimental Cell Research 313(13):2833-2844 (2007).
[19] Usami N, Ishikura S, Abe H, Nagano M, Uebuchi M, Kuniyasu A, Otagiri M, Nakayama H, Imamura Y, Hara A(2003). Cloning, expression and tissue distribution of a tetrameric form of pig carbonyl reductase. Chem Biol Intreact. 1:353-361
[20] Kallberg Y, Oppermann U, Nvall HJ, Persson B (2002) Short-chain dehydrogenase/ reductase(SDR) relationship: A large family with eight clusters common to human, animal, and plant genomes. Protein Sci 11:636-641.
[21] Oppermann U, Filling C, Hult M, Shafqat N, Wu X, Lindh M, Nordling E (2003)Short-chain dehydrogenase/reductase(SDR): the 2002 update. Chem Biol Interact 1:247-253.
[22] Joseph MJ, Milanie JB, Brian PC, Mitchell L, Trevor MP (1997) Review article: Comparative anatomy of the aldo-keto reductase superfamily. Biochem J 326:625-636.
[23] Kallberg Y, Oppermann U, Jornvall H, Persson B (2002) Short-chain dehydrogenase/ reductase(SDRs) coenzyme-based functional assignments in completed genomes. Eur J Biochem 269:4409-4417.
[24] Grabowski PJ, Black DL. Alternative RNA splicing in the nervous system. Prog Neurobiol 2001;65:289–308.
[25] Venables JP. Alternative splicing in the testes. Curr Opin Genet Dev 2002;12:615–9
[26] International human genome sequencing consortium(2004): Finishing the euchromatic sequence of the human genome. Nature 431:931-945.
[27] Black DL(2000) Protein diversity from alternative splicing: A challenge for bioinformatics and post-genome biology. Cell 103:367-370.
[28] Faustino NA, Cooper TA. Pre-mRNA splicing and human disease[J]. Genes Dev 2003,17(4):419-437.
[29] Garcia-Blanco MA, Baraniak AP, Lasda EL. Alternative splicing in disease and therapy[J]. Nat Biotechnol 2004,22(5):535-546.
[30] Qiang Xu, Christopher Lee. Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences[J]. Nucleic Acids Research 2003,31(19):5635-5643.
[31] Wang Z, Lo HS, Yang H, et al. Computational analysis and experimental validation of tumorassociated alternative RNA splicing in human cancer[J]. Cancer Res 2003,63(3):655-657.
[32] Hu, A. and Fu, X.D. (2007) Splicing oncogenes. Nat Struct Mol Biol, 14, 174-175.
[33] Black, D.L. (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem,72, 291-336.
[34] Maniatis, T. and Tasic, B. (2002) Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature, 418, 236-243.
[35] Song XH, Liang B, Liu GF, Li R., Xie JP, Du K and Huang DY. Expression of a novel alternatively spliced variant of NADP(H)-dependent retinol dehydrogenase/ reductase with deletion of exon 3 in cervical squamous carcinoma. International Journal of Cancer 2007,120:1618 -1626.
[36]李一凡,刘戈飞,宋旭红等.一种人神经母细胞瘤辅酶II-依赖性视黄醇脱氢酶cDNA的克隆及特征分析.癌变畸变突变,2005,17(6):321-326.
[37]宋旭红,梁斌,刘戈飞,李蕊,谢建平,黄东阳宫颈鳞癌组织中NRDR选择性剪切新亚型的鉴定及意义肿瘤2006,26(12):1088-1092.
[38] Wang Y, Leung FC (2004) An evaluation of new criteria for CpG islands in the human genome as gene markers. Bioinformatics 20:1170-1177
[39] Yanagisawa K, Tago K, Hayakawa M, Ohki M, Iwahana H, Tominaga S . A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK). Biochem J 15:159-166
[2] Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J. 1996,10:940-954.
[3] Mangelsdorf DJ, Thummel C, Beato M. The nuclear receptor superfamily: the second decade. Cell 1995,83:835-839.
[4] Altucci L, Gronemeyer H. Nuclear receptors in cell life and death. Trends Endocrinol. Metab. 2001,12:460-468
[5] Boulogne B, Levacher C, Durand P. Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: Immnolocalization and implication in the control of the number of gonocytes. Biol. Reprod. 1999,61:1548-1557.
[6] Mendelsohn C, Lohnes D, Decimo D. Function of the retinoic acid receptors (RARs) during development(II).Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development. 1994,120:2749-2771.
[7] Malpel S, Mendelsohn C, Cardoso WV. Regulation of retinoic acid signaling during lung morphogenesis. Development 2000,127:3057-3067.
[8] Huang, M.E., Ye, Y-C., Chen, S-R., Chai, J-R.,Lu, J-X., Shoa, Lin, Guo, L-J., and Wang, Z-Y. (1988) Use of all-transretinoic acid in the treatment of acute promyelocytic leukemia.Blood 72, 567-572.
[9] Colucciello M. Pseudotumor cerebri induced by all-trans retinoic acid treatment of acute promyelocytic leukemia. Arch Ophthalmol. 2003,121: 1064-1069.
[10] Recchia F, Saggio G, Nuzzo A, Biondi E, Di-Blasio A, Cesta A, Candeloro G, Alesse E, Rea S. Multicenter phase 2 study of interleukin-2 and 13-cis retinoic acid as maintenance therapy in advanced non-small-cell lung cancer. J Immunother. 2006, 29: 87-94.
[11] Brtko J, Thalhamer J. Renaissance of the biologically active vitamin A derivatives: established and novel directed therapies for cancer and chemoprevention. Curr Pham Des 2003, 9: 2067-2077.
[12] Grubbs CJ, Hill DL, Bland KI, Beenken SW, Lin TH, Eto I, Atigadda VR, Vines KK, Brouillette WJ, Muccio DD. 9cUAB30, an RXR specific retinoid, and/or tamoxifen in the prevention of methylnitrosourea-induced mammary cancers. Cancer Lett. 2003,201: 17-24.
[13] Kannan K, Khan HA, Jain R, Hussein SS, Dennison D. All-trans retinoic acid-induced myositis. Br J Haematol 2005,131: 560-569.
[14] Zhong JC, Huang DY, Liu GF, Jin HY,Yang YM, Li YF, Song XH, Du K. Effect of all-trans retinoic acid on orphan receptor APJ signaling in spontaneously hypertensive rats. Cardiovascular Res 65(3): 743-750 (2005).
[15] JC Zhong, DY Huang, YM Yang, YF Li, GF Liu, XH Song, K Du. Upregulation of angiotensin-converting enzyme 2 by all-trans retinoic acid in spontaneously hypertensive rats. Hypertension 44:907-912 (2004).
[16] Yan-Mei Yang, Dong-Yang Huang, Ge-Fei Liu, Jiu-Chang Zhong, Yi-Fan Li, Xu-Hong Song. Inhibitory Effects of Vitamin A on TCDD-induced Cytochrome P-450 1A1 Enzyme Activity and Expression. Toxicological Sciences 85:727-734 (2005).
[17] Yan-Mei Yang, Dong-Yang Huang, Ge-Fei Liu, Jiu-Chang Zhong, Yi-Fan Li, Xu-Hong Song. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on vitamin A metabolism in mice. J Biochem Molecular Toxicology 19:327-335 (2005).
[18] Liang B, Song XH, Liu GF, Li R, Xie JP, Xiao LF, Du MD, Zhang QX,Xu XY, Gan XQ, Huang DY. Involvement of TR3/Nur77 translocation to the endoplasmic reticulum in ER stress-induced apoptosis. Experimental Cell Research 313(13):2833-2844 (2007).
[19] Usami N, Ishikura S, Abe H, Nagano M, Uebuchi M, Kuniyasu A, Otagiri M, Nakayama H, Imamura Y, Hara A(2003). Cloning, expression and tissue distribution of a tetrameric form of pig carbonyl reductase. Chem Biol Intreact. 1:353-361
[20] Kallberg Y, Oppermann U, Nvall HJ, Persson B (2002) Short-chain dehydrogenase/ reductase(SDR) relationship: A large family with eight clusters common to human, animal, and plant genomes. Protein Sci 11:636-641.
[21] Oppermann U, Filling C, Hult M, Shafqat N, Wu X, Lindh M, Nordling E (2003)Short-chain dehydrogenase/reductase(SDR): the 2002 update. Chem Biol Interact 1:247-253.
[22] Joseph MJ, Milanie JB, Brian PC, Mitchell L, Trevor MP (1997) Review article: Comparative anatomy of the aldo-keto reductase superfamily. Biochem J 326:625-636.
[23] Kallberg Y, Oppermann U, Jornvall H, Persson B (2002) Short-chain dehydrogenase/ reductase(SDRs) coenzyme-based functional assignments in completed genomes. Eur J Biochem 269:4409-4417.
[24] Grabowski PJ, Black DL. Alternative RNA splicing in the nervous system. Prog Neurobiol 2001;65:289–308.
[25] Venables JP. Alternative splicing in the testes. Curr Opin Genet Dev 2002;12:615–9
[26] International human genome sequencing consortium(2004): Finishing the euchromatic sequence of the human genome. Nature 431:931-945.
[27] Black DL(2000) Protein diversity from alternative splicing: A challenge for bioinformatics and post-genome biology. Cell 103:367-370.
[28] Faustino NA, Cooper TA. Pre-mRNA splicing and human disease[J]. Genes Dev 2003,17(4):419-437.
[29] Garcia-Blanco MA, Baraniak AP, Lasda EL. Alternative splicing in disease and therapy[J]. Nat Biotechnol 2004,22(5):535-546.
[30] Qiang Xu, Christopher Lee. Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences[J]. Nucleic Acids Research 2003,31(19):5635-5643.
[31] Wang Z, Lo HS, Yang H, et al. Computational analysis and experimental validation of tumorassociated alternative RNA splicing in human cancer[J]. Cancer Res 2003,63(3):655-657.
[32] Hu, A. and Fu, X.D. (2007) Splicing oncogenes. Nat Struct Mol Biol, 14, 174-175.
[33] Black, D.L. (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem,72, 291-336.
[34] Maniatis, T. and Tasic, B. (2002) Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature, 418, 236-243.
[35] Song XH, Liang B, Liu GF, Li R., Xie JP, Du K and Huang DY. Expression of a novel alternatively spliced variant of NADP(H)-dependent retinol dehydrogenase/ reductase with deletion of exon 3 in cervical squamous carcinoma. International Journal of Cancer 2007,120:1618 -1626.
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