利用外显子捕获测序技术筛选及验证肝癌相关突变基因
摘要
肝细胞癌(HCC)是世界范围内最常见的癌症之一,其具有浸润和转移的遗传倾向性。实验室前期采用外显子捕获测序技术,利用Illumina Solexa测序平台分析鉴定伴发有门静脉癌栓(PVTT)的10位肝细胞癌病人样本的体细胞突变。PVTT是肝细胞癌肝内转移形式。对10例肝癌病人的癌旁组织、原位癌及门静脉癌栓共29个组织样本(其中一例病人癌栓组织样本降解)的Solexa测序结果进行分析发现在10例肝癌病人的癌组织中有122574个潜在的突变位点。通过设定特定的筛选条件进行筛选过滤,我们选取了381个定位于编码外显子区域的非沉默性突变位点,通过PCR-Sanger测序法进一步验证,最终重新验证了165个非沉默性突变,验证率为43%。在经过验证的165个非沉默突变位点中经常出现碱基的转换和颠换。我们采用RNA干扰的方法评价了91个突变基因对于细胞活性的影响。结果显示,TMEM35、CDK14、HOXA1、ABCA1都是潜在的癌基因,赋予了肝癌细胞生长和浸润的能力。进一步研究发现TMEM35基因编码具有四跨膜结构域的膜蛋白,其在肝癌细胞生长中发挥重要作用。
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and exhibits an inherited predisposition to infiltrate and metastasize. Here, we use exomic sequencing parallel sequences on an Illumina Solexa platform to identify the somatic mutations in ten HCC patients with portal vein tumor thrombus (PVTT), the intrahepatic metastasis. Through sequencing 29 samples of ten HCC patients'non-cancerous livers, primary and PVTT (one of PVTTwas degradation) we found 122574 somatic mutations on ten HCC patients with PVTT.After analyzed according to described method, we selected 381 non-silenced mutations which located on exon for futher test by sanger sequencing. Finally we retested 165 non-silenced mutations, validation rate was 43%. Base transitions and base transversions were frequently found among 165 confirmed non-silenced mutations. We assessed the role of 91 mutated genes in cellular survival via RNA interference; the results suggest that TMEM35, CDK14, HOXA1, ABCA1 could be potential cancer genes that confer growth and infiltration capabilities to HCC cells. For futher study we found TMEM35 gene, which encode a tetraspanin, have an important role in cell growth of hepatoma cells.
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and exhibits an inherited predisposition to infiltrate and metastasize. Here, we use exomic sequencing parallel sequences on an Illumina Solexa platform to identify the somatic mutations in ten HCC patients with portal vein tumor thrombus (PVTT), the intrahepatic metastasis. Through sequencing 29 samples of ten HCC patients'non-cancerous livers, primary and PVTT (one of PVTTwas degradation) we found 122574 somatic mutations on ten HCC patients with PVTT.After analyzed according to described method, we selected 381 non-silenced mutations which located on exon for futher test by sanger sequencing. Finally we retested 165 non-silenced mutations, validation rate was 43%. Base transitions and base transversions were frequently found among 165 confirmed non-silenced mutations. We assessed the role of 91 mutated genes in cellular survival via RNA interference; the results suggest that TMEM35, CDK14, HOXA1, ABCA1 could be potential cancer genes that confer growth and infiltration capabilities to HCC cells. For futher study we found TMEM35 gene, which encode a tetraspanin, have an important role in cell growth of hepatoma cells.
引文
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[21]Zeggini, E., et al., Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes. Nat Genet,2008.40(5):p.638-45.
[22]Altshuler, D., M. J. Daly, and E. S. Lander, Genetic mapping in human disease. Science,2008.322(5903):p.881-8.
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[27]Jones, S., et al., Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science,2008.321 (5897):p.1801-6.
[28]Ding, L., et al., Somatic mutations affect key pathways in lung adenocarcinoma. Nature,2008.455(7216):p.1069-75.
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[30]Jones, S., et al., Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Science,2010.330(6001):p.228-31.
[31]Wiegand, K. C., et al., ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Med,2010.363(16):p.1532-43.
[32]Li, Y., et al., Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol,2001.7(5):p.630-6.
[33]Boutros, M., L. P. Bras, and W. Huber, Analysis of cell-based RNAi screens. Genome Biol,2006.7(7):p. R66.
[34]Wittwer, C. T., et al., Real-time multiplex PCR assays. Methods,2001. 25(4):p.430-42.
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[36]Yuan, J. S., D. Wang, and C. N. Stewart, Jr., Statistical methods for efficiency adjusted real-time PCR quantification. Biotechnol J,2008. 3(1):p.112-23.
[37]Livak, K. J. and T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods,2001.25(4):p.402-8.
[38]Cantor, S.B., et al., BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Cell,2001. 105(1):p.149-60.
[39]Levran,O., et al., The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia. Nat Genet,2005.37(9):p.931-3.
[40]Mao, Y., et al., Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell,2009.136(6):p.1017-31.
[41]Hattori, T., et al., DISC1 regulates cell-cell adhesion, cell-matrix adhesion and neurite outgrowth. Mol Psychiatry,2010.15(8):p.778, 798-809.
[42]Wells, C.M. and G. E. Jones, The emerging importance of group II PAKs. Biochem J,2010.425(3):p.465-73.
[43]Shuker, N., et al., ATP-binding cassette transporters as pharmacogenetic biomarkers for kidney transplantation. Clin Chim Acta,2011.
[44]Chiba, S., Homeobox genes in normal hematopoiesis and leukemogenesis. Int J Hematol,1998.68(4):p.343-53.
[45]Lawrence, H.J., et al., Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood,1997.89(6):p.1922-30.
[46]Castronovo, V., et al., Homeobox genes:potential candidates for the transcriptional control of the transformed and invasive phenotype. Biochem Pharmacol,1994.47(1):p.137-43.
[47]Thirman, M. J., et al., Cloning of ELL, a gene that fuses to MLL in a t (11; 19) (q23;p13.1) in acute myeloid leukemia. Proc Natl Acad Sci U S A, 1994.91(25):p.12110-4.
[48]Mitani, K., et al., Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11; 19) (q23;p13.1) translocation. Blood,1995. 85(8):p.2017-24.
[49]Hahn, J., et al., Apoptosis induction and growth suppression by U19/Eaf2 is mediated through its ELL-binding domain. Prostate,2007.67(2):p. 146-53.
[50]Johnstone, R. W., et al., Functional analysis of the leukemia protein ELL: evidence for a role in the regulation of cell growth and survival. Mol Cell Biol,2001.21(5):p.1672-81.
[51]Shilatifard, A., et al., An RNA polymerase Ⅱ elongation factor encoded by the human ELL gene. Science,1996.271(5257):p.1873-6.
[52]Pascual-Le Tallec, L., et al., The elongation factor ELL (eleven-nineteen lysine-rich leukemia) is a selective coregulator for steroid receptor functions. Mol Endocrinol,2005.19(5):p.1158-69.
[53]Mitani, K., et al., Nonredundant roles of the elongation factor MEN in postimplantation development. Biochem Biophys Res Commun,2000.279(2): p.563-7.
[54]Tran, P. V., M. K. Georgieff, and W. C. Engeland, Sodium depletion increases sympathetic neurite outgrowth and expression of a novel TMEM35 gene-derived protein (TUF1) in the rat adrenal zona glomerulosa. Endocrinology,2010.151(10):p.4852-60.
[55]Juretic, D., L. Zoranic, and D. Zucic, Basic charge clusters and predictions of membrane protein topology. J Chem Inf Comput Sci,2002. 42(3):p.620-32.
[56]Greenman, C., et al., Patterns of somatic mutation in human cancer genomes. Nature,2007.446(7132):p.153-8.
[57]Hussain, S.P., et al., TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer. Oncogene, 2007.26(15):p.2166-76.
[58]Aguilar, F., S. P. Hussain, and P. Cerutti, Aflatoxin B1 induces the transversion of G-->T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc Natl Acad Sci U S A,1993.90(18):p.8586-90.
[59]Hsu, I.C., et al., Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature,1991.350(6317):p.427-8.
[60]Borland, G., et al., Microtubule-associated protein 1B-light chain 1 enhances activation of Rapl by exchange protein activated by cyclic AMP but not intracellular targeting. Mol Pharmacol,2006.69(1):p.374-84.
[61]Cabodi, S., et al., Integrin signalling adaptors:not only figurants in the cancer story. Nat Rev Cancer,2010.10(12):p.858-70.
[62]Iyer, A., et al., Different cytokeratin and neuronal cell adhesion molecule staining patterns in focal nodular hyperplasia and hepatic adenoma and their significance. Hum Pathol,2008.39(9):p.1370-7.
[63]Ke, A. W., et al., CD151 amplifies signaling by integrin alpha6betal to PI3K and induces the epithelial-mesenchymal transition in HCC cells. Gastroenterology,2011.140(5):p.1629-41 e15.
[64]Sadej, R., et al., CD151 regulates tumorigenesis by modulating the communication between tumor cells and endothelium. Mol Cancer Res,2009. 7(6):p.787-98.
[65]Ke, A. W., et al., Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma. Hepatology,2009.49(2): p.491-503.
[2]Badrinath, P. and S. Ramaiah, Trends in primary liver cancer. Lancet,1998. 351(9097):p.215.
[3]Donato, F., P. Boffetta, and M. Puoti, A meta-analysis of epidemiological studies on the combined effect of hepatitis B and C virus infections in causing hepatocellular carcinoma. Int J Cancer,1998.75(3):p.347-54.
[4]Primary liver cancer in Japan. Clinicopathologic features and results of surgical treatment. Liver Cancer Study Group of Japan. Ann Surg,1990. 211(3):p.277-87.
[5]Llovet, J. M., et al., Natural history of untreated nonsurgical hepatocellular carcinoma:rationale for the design and evaluation of therapeutic trials. Hepatology,1999.29(1):p.62-7.
[6]Staib, F., et al., TP53 and liver carcinogenesis. Hum Mutat,2003.21(3): p.201-16.
[7]de La Coste, A., et al., Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci U S A,1998.95(15):p.8847-51.
[8]Dulbecco, R., A turning point in cancer research:sequencing the human genome. Science,1986.231(4742):p.1055-6.
[9]Ng, S. B., et al., Targeted capture and massively parallel sequencing of 12 human exomes. Nature,2009.461 (7261):p.272-6.
[10]Choi, M., et al., Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci U S A,2009.106(45):p. 19096-101.
[11]Parsons, D.W., et al., An integrated genomic analysis of human glioblastoma multiforme. Science,2008.321(5897):p.1807-12.
[12]Goldberg, M.L., et al., Isolation of specific RNA's using DNA covalently linked to diazobenzyloxymethyl cellulose or paper. Methods Enzymol,1979. 68:p.206-20.
[13]Albert, T. J., et al., Direct selection of human genomic loci by microarray hybridization. Nat Methods,2007.4(11):p.903-5.
[14]Ng, S.B., et al., Exome sequencing identifies the cause of a mendelian disorder. Nat Genet,2010.42(1):p.30-5.
[15]Margulies, M., et al., Genome sequencing in microfabricated high-density picolitre reactors. Nature,2005.437(7057):p.376-80.
[16]Bentley, D. R., et al., Accurate whole human genome sequencing using reversible terminator chemistry. Nature,2008.456(7218):p.53-9.
[17]Smith, D. R., et al., Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Res,2008.18(10):p. 1638-42.
[18]Cooper, D. N., P. D. Stenson, and N. A. Chuzhanova, The Human Gene Mutation Database (HGMD) and its exploitation in the study of mutational mechanisms. Curr Protoc Bioinformatics,2006. Chapter 1:p. Unit 1 13.
[19]Aulchenko, Y. S., et al., Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nat Genet,2009.41(1): p.47-55.
[20]Kathiresan, S., et al., Common variants at 30 loci contribute to polygenic dyslipidemia. Nat Genet,2009.41(1):p.56-65.
[21]Zeggini, E., et al., Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes. Nat Genet,2008.40(5):p.638-45.
[22]Altshuler, D., M. J. Daly, and E. S. Lander, Genetic mapping in human disease. Science,2008.322(5903):p.881-8.
[23]Hirschhorn, J.N., Genomewide association studies--illuminating biologic pathways. N Engl J Med,2009.360(17):p.1699-701.
[24]Stratton, M. R., P. J. Campbell, and P. A. Futreal, The cancer genome. Nature, 2009.458(7239):p.719-24.
[25]Wood, L. D., et al., The genomic landscapes of human breast and colorectal cancers. Science,2007.318(5853):p.1108-13.
[26]Sjoblom, T., et al., The consensus coding sequences of human breast and colorectal cancers. Science,2006.314(5797):p.268-74.
[27]Jones, S., et al., Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science,2008.321 (5897):p.1801-6.
[28]Ding, L., et al., Somatic mutations affect key pathways in lung adenocarcinoma. Nature,2008.455(7216):p.1069-75.
[29]Dalgliesh, G. L., et al., Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature,2010.463(7279):p. 360-3.
[30]Jones, S., et al., Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Science,2010.330(6001):p.228-31.
[31]Wiegand, K. C., et al., ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Med,2010.363(16):p.1532-43.
[32]Li, Y., et al., Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol,2001.7(5):p.630-6.
[33]Boutros, M., L. P. Bras, and W. Huber, Analysis of cell-based RNAi screens. Genome Biol,2006.7(7):p. R66.
[34]Wittwer, C. T., et al., Real-time multiplex PCR assays. Methods,2001. 25(4):p.430-42.
[35]Giulietti, A., et al., An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods,2001.25(4): p.386-401.
[36]Yuan, J. S., D. Wang, and C. N. Stewart, Jr., Statistical methods for efficiency adjusted real-time PCR quantification. Biotechnol J,2008. 3(1):p.112-23.
[37]Livak, K. J. and T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods,2001.25(4):p.402-8.
[38]Cantor, S.B., et al., BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Cell,2001. 105(1):p.149-60.
[39]Levran,O., et al., The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia. Nat Genet,2005.37(9):p.931-3.
[40]Mao, Y., et al., Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell,2009.136(6):p.1017-31.
[41]Hattori, T., et al., DISC1 regulates cell-cell adhesion, cell-matrix adhesion and neurite outgrowth. Mol Psychiatry,2010.15(8):p.778, 798-809.
[42]Wells, C.M. and G. E. Jones, The emerging importance of group II PAKs. Biochem J,2010.425(3):p.465-73.
[43]Shuker, N., et al., ATP-binding cassette transporters as pharmacogenetic biomarkers for kidney transplantation. Clin Chim Acta,2011.
[44]Chiba, S., Homeobox genes in normal hematopoiesis and leukemogenesis. Int J Hematol,1998.68(4):p.343-53.
[45]Lawrence, H.J., et al., Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood,1997.89(6):p.1922-30.
[46]Castronovo, V., et al., Homeobox genes:potential candidates for the transcriptional control of the transformed and invasive phenotype. Biochem Pharmacol,1994.47(1):p.137-43.
[47]Thirman, M. J., et al., Cloning of ELL, a gene that fuses to MLL in a t (11; 19) (q23;p13.1) in acute myeloid leukemia. Proc Natl Acad Sci U S A, 1994.91(25):p.12110-4.
[48]Mitani, K., et al., Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11; 19) (q23;p13.1) translocation. Blood,1995. 85(8):p.2017-24.
[49]Hahn, J., et al., Apoptosis induction and growth suppression by U19/Eaf2 is mediated through its ELL-binding domain. Prostate,2007.67(2):p. 146-53.
[50]Johnstone, R. W., et al., Functional analysis of the leukemia protein ELL: evidence for a role in the regulation of cell growth and survival. Mol Cell Biol,2001.21(5):p.1672-81.
[51]Shilatifard, A., et al., An RNA polymerase Ⅱ elongation factor encoded by the human ELL gene. Science,1996.271(5257):p.1873-6.
[52]Pascual-Le Tallec, L., et al., The elongation factor ELL (eleven-nineteen lysine-rich leukemia) is a selective coregulator for steroid receptor functions. Mol Endocrinol,2005.19(5):p.1158-69.
[53]Mitani, K., et al., Nonredundant roles of the elongation factor MEN in postimplantation development. Biochem Biophys Res Commun,2000.279(2): p.563-7.
[54]Tran, P. V., M. K. Georgieff, and W. C. Engeland, Sodium depletion increases sympathetic neurite outgrowth and expression of a novel TMEM35 gene-derived protein (TUF1) in the rat adrenal zona glomerulosa. Endocrinology,2010.151(10):p.4852-60.
[55]Juretic, D., L. Zoranic, and D. Zucic, Basic charge clusters and predictions of membrane protein topology. J Chem Inf Comput Sci,2002. 42(3):p.620-32.
[56]Greenman, C., et al., Patterns of somatic mutation in human cancer genomes. Nature,2007.446(7132):p.153-8.
[57]Hussain, S.P., et al., TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer. Oncogene, 2007.26(15):p.2166-76.
[58]Aguilar, F., S. P. Hussain, and P. Cerutti, Aflatoxin B1 induces the transversion of G-->T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc Natl Acad Sci U S A,1993.90(18):p.8586-90.
[59]Hsu, I.C., et al., Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature,1991.350(6317):p.427-8.
[60]Borland, G., et al., Microtubule-associated protein 1B-light chain 1 enhances activation of Rapl by exchange protein activated by cyclic AMP but not intracellular targeting. Mol Pharmacol,2006.69(1):p.374-84.
[61]Cabodi, S., et al., Integrin signalling adaptors:not only figurants in the cancer story. Nat Rev Cancer,2010.10(12):p.858-70.
[62]Iyer, A., et al., Different cytokeratin and neuronal cell adhesion molecule staining patterns in focal nodular hyperplasia and hepatic adenoma and their significance. Hum Pathol,2008.39(9):p.1370-7.
[63]Ke, A. W., et al., CD151 amplifies signaling by integrin alpha6betal to PI3K and induces the epithelial-mesenchymal transition in HCC cells. Gastroenterology,2011.140(5):p.1629-41 e15.
[64]Sadej, R., et al., CD151 regulates tumorigenesis by modulating the communication between tumor cells and endothelium. Mol Cancer Res,2009. 7(6):p.787-98.
[65]Ke, A. W., et al., Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma. Hepatology,2009.49(2): p.491-503.