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Published in: BMC Cancer 1/2013

Open Access 01-12-2013 | Research article

Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3as a novel lung cancer-related gene

Authors: Mingsong Wu, Tao Tu, Yunchao Huang, Yi Cao

Published in: BMC Cancer | Issue 1/2013

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Abstract

Background

To understand the carcinogenesis caused by accumulated genetic and epigenetic alterations and seek novel biomarkers for various cancers, studying differentially expressed genes between cancerous and normal tissues is crucial. In the study, two cDNA libraries of lung cancer were constructed and screened for identification of differentially expressed genes.

Methods

Two cDNA libraries of differentially expressed genes were constructed using lung adenocarcinoma tissue and adjacent nonmalignant lung tissue by suppression subtractive hybridization. The data of the cDNA libraries were then analyzed and compared using bioinformatics analysis. Levels of mRNA and protein were measured by quantitative real-time polymerase chain reaction (q-RT-PCR) and western blot respectively, as well as expression and localization of proteins were determined by immunostaining. Gene functions were investigated using proliferation and migration assays after gene silencing and gene over-expression.

Results

Two libraries of differentially expressed genes were obtained. The forward-subtracted library (FSL) and the reverse-subtracted library (RSL) contained 177 and 59 genes, respectively. Bioinformatic analysis demonstrated that these genes were involved in a wide range of cellular functions. The vast majority of these genes were newly identified to be abnormally expressed in lung cancer. In the first stage of the screening for 16 genes, we compared lung cancer tissues with their adjacent non-malignant tissues at the mRNA level, and found six genes (ERGIC3, DDR1, HSP90B1, SDC1, RPSA, and LPCAT1) from the FSL were significantly up-regulated while two genes (GPX3 and TIMP3) from the RSL were significantly down-regulated (P < 0.05). The ERGIC3 protein was also over-expressed in lung cancer tissues and cultured cells, and expression of ERGIC3 was correlated with the differentiated degree and histological type of lung cancer. The up-regulation of ERGIC3 could promote cellular migration and proliferation in vitro.

Conclusions

The two libraries of differentially expressed genes may provide the basis for new insights or clues for finding novel lung cancer-related genes; several genes were newly found in lung cancer with ERGIC3 seeming a novel lung cancer-related gene. ERGIC3 may play an active role in the development and progression of lung cancer.
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Literature
1.
go back to reference Risch A, Plass C: Lung cancer epigenetics and genetics. Int J Cancer. 2008, 101: 1-7.CrossRef Risch A, Plass C: Lung cancer epigenetics and genetics. Int J Cancer. 2008, 101: 1-7.CrossRef
2.
go back to reference Nindl I, Dang C, Forschner T, Kuban RJ, Meyer T, Sterry W, Stockfleth E: Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling. Mol Cancer. 2006, 5: 30-10.1186/1476-4598-5-30.CrossRefPubMedPubMedCentral Nindl I, Dang C, Forschner T, Kuban RJ, Meyer T, Sterry W, Stockfleth E: Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling. Mol Cancer. 2006, 5: 30-10.1186/1476-4598-5-30.CrossRefPubMedPubMedCentral
3.
go back to reference Selvarajah GT, Kirpensteijn J, van Wolferen ME, Rao NA, Fieten H, Mol JA: Gene expression profiling of canine osteosarcoma reveals genes associated with short and long survival times. Mol Cancer. 2009, 8: 72-10.1186/1476-4598-8-72.CrossRefPubMedPubMedCentral Selvarajah GT, Kirpensteijn J, van Wolferen ME, Rao NA, Fieten H, Mol JA: Gene expression profiling of canine osteosarcoma reveals genes associated with short and long survival times. Mol Cancer. 2009, 8: 72-10.1186/1476-4598-8-72.CrossRefPubMedPubMedCentral
4.
go back to reference Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD: Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA. 1996, 93: 6025-6030. 10.1073/pnas.93.12.6025.CrossRefPubMedPubMedCentral Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD: Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA. 1996, 93: 6025-6030. 10.1073/pnas.93.12.6025.CrossRefPubMedPubMedCentral
5.
go back to reference Yang ZJ, Peng ZS, Yang H, Yang J, Wei SH, Cai P: Suppression subtractive hybridization identified differentially expressed genes in pistil mutations in wheat. Plant Mol Biol Rep. 2011, 29: 431-439. 10.1007/s11105-010-0249-2.CrossRef Yang ZJ, Peng ZS, Yang H, Yang J, Wei SH, Cai P: Suppression subtractive hybridization identified differentially expressed genes in pistil mutations in wheat. Plant Mol Biol Rep. 2011, 29: 431-439. 10.1007/s11105-010-0249-2.CrossRef
6.
go back to reference Petroziello J, Yamane A, Westendorf L, Thompson M, McDonagh C, Cerveny C, Law CL, Wahl A, Carter P: Suppression subtractive hybridization and expression profiling identifies a unique set of genes overexpressed in non-small-cell lung cancer. Oncogene. 2004, 23: 7734-7745. 10.1038/sj.onc.1207921.CrossRefPubMed Petroziello J, Yamane A, Westendorf L, Thompson M, McDonagh C, Cerveny C, Law CL, Wahl A, Carter P: Suppression subtractive hybridization and expression profiling identifies a unique set of genes overexpressed in non-small-cell lung cancer. Oncogene. 2004, 23: 7734-7745. 10.1038/sj.onc.1207921.CrossRefPubMed
7.
go back to reference Difilippantonio S, Chen Y, Pietas A, Schlüns K, Pacyna-Gengelbach M, Deutschmann N, Padilla-Nash H, Ried T, Petersen I: Gene expression profiles in human non-small and small-cell lung cancers. Eur J Cancer. 2003, 39: 1936-1947. 10.1016/S0959-8049(03)00419-2.CrossRefPubMed Difilippantonio S, Chen Y, Pietas A, Schlüns K, Pacyna-Gengelbach M, Deutschmann N, Padilla-Nash H, Ried T, Petersen I: Gene expression profiles in human non-small and small-cell lung cancers. Eur J Cancer. 2003, 39: 1936-1947. 10.1016/S0959-8049(03)00419-2.CrossRefPubMed
8.
go back to reference Sun W, Zhang K, Zhang X, Lei W, Xiao T, Ma J, Guo S, Shao S, Zhang H, Liu Y, Yuan J, Hu Z, Ma Y, Feng X, Hu S, Zhou J, Cheng S, Gao Y: Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization. Cancer Lett. 2004, 212: 83-93. 10.1016/j.canlet.2004.03.023.CrossRefPubMed Sun W, Zhang K, Zhang X, Lei W, Xiao T, Ma J, Guo S, Shao S, Zhang H, Liu Y, Yuan J, Hu Z, Ma Y, Feng X, Hu S, Zhou J, Cheng S, Gao Y: Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization. Cancer Lett. 2004, 212: 83-93. 10.1016/j.canlet.2004.03.023.CrossRefPubMed
9.
go back to reference Liu Y, Sun W, Zhang K, Zheng H, Ma Y, Lin D, Zhang X, Feng L, Lei W, Zhang Z, Guo S, Han N, Tong W, Feng X, Gao Y, Cheng S: Identification of genes differentially expressed in human primary lung squamous cell carcinoma. Lung Cancer. 2007, 56: 307-317. 10.1016/j.lungcan.2007.01.016.CrossRefPubMed Liu Y, Sun W, Zhang K, Zheng H, Ma Y, Lin D, Zhang X, Feng L, Lei W, Zhang Z, Guo S, Han N, Tong W, Feng X, Gao Y, Cheng S: Identification of genes differentially expressed in human primary lung squamous cell carcinoma. Lung Cancer. 2007, 56: 307-317. 10.1016/j.lungcan.2007.01.016.CrossRefPubMed
10.
go back to reference Bangur CS, Switzer A, Fan L, Marton MJ, Meyer MR, Wang T: Identification of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis. Oncogene. 2002, 21: 3814-3825. 10.1038/sj.onc.1205480.CrossRefPubMed Bangur CS, Switzer A, Fan L, Marton MJ, Meyer MR, Wang T: Identification of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis. Oncogene. 2002, 21: 3814-3825. 10.1038/sj.onc.1205480.CrossRefPubMed
11.
go back to reference Orci L, Ravazzola M, Mack GJ, Barlowe C, Otte S: Mammalian Erv46 localizes to the endoplasmic reticulum-Golgi intermediate compartment and to cis-Golgi cisternae. Proc Natl Acad Sci USA. 2003, 100: 4586-4591. 10.1073/pnas.0730885100.CrossRefPubMedPubMedCentral Orci L, Ravazzola M, Mack GJ, Barlowe C, Otte S: Mammalian Erv46 localizes to the endoplasmic reticulum-Golgi intermediate compartment and to cis-Golgi cisternae. Proc Natl Acad Sci USA. 2003, 100: 4586-4591. 10.1073/pnas.0730885100.CrossRefPubMedPubMedCentral
12.
go back to reference Brambilla E, Travis WD, Colby TV, Corrin B, Shimosato Y: The new world health organization classification of lung tumours. Eur Respir J. 2001, 18: 1059-1068. 10.1183/09031936.01.00275301.CrossRefPubMed Brambilla E, Travis WD, Colby TV, Corrin B, Shimosato Y: The new world health organization classification of lung tumours. Eur Respir J. 2001, 18: 1059-1068. 10.1183/09031936.01.00275301.CrossRefPubMed
13.
go back to reference Sobin LH, Wittekind C: Lung. International union against cancer [UICC]. TNM classification of malignant tumours. Edited by: Sobin LH, Wittekind C. 1997, Baltimore, MD: Wiley-Liss, 93-97. Sobin LH, Wittekind C: Lung. International union against cancer [UICC]. TNM classification of malignant tumours. Edited by: Sobin LH, Wittekind C. 1997, Baltimore, MD: Wiley-Liss, 93-97.
14.
go back to reference Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008, 3: 1101-1108. 10.1038/nprot.2008.73.CrossRefPubMed Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008, 3: 1101-1108. 10.1038/nprot.2008.73.CrossRefPubMed
15.
go back to reference Cao Y, Merling A, Crocker PR, Keller R, Schwartz-Albiez R: Differential expression of β-galactoside α2, 6 sialyltransferase and sialoglycans in normal and cirrhotic liver and hepatocellular carcinoma. Lab Invest. 2002, 82: 1515-1524.CrossRefPubMed Cao Y, Merling A, Crocker PR, Keller R, Schwartz-Albiez R: Differential expression of β-galactoside α2, 6 sialyltransferase and sialoglycans in normal and cirrhotic liver and hepatocellular carcinoma. Lab Invest. 2002, 82: 1515-1524.CrossRefPubMed
16.
go back to reference Sjöblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N, Szabo S, Buckhaults P, Farrell C, Meeh P, Markowitz SD, Willis J, Dawson D, Willson JK, Gazdar AF, Hartigan J, Wu L, Liu C, Parmigiani G, Park BH, Bachman KE, Papadopoulos N, Vogelstein B, Kinzler KW, Velculescu VE: The consensus coding sequences of human breast and colorectal cancers. Science. 2006, 314: 268-274. 10.1126/science.1133427.CrossRefPubMed Sjöblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N, Szabo S, Buckhaults P, Farrell C, Meeh P, Markowitz SD, Willis J, Dawson D, Willson JK, Gazdar AF, Hartigan J, Wu L, Liu C, Parmigiani G, Park BH, Bachman KE, Papadopoulos N, Vogelstein B, Kinzler KW, Velculescu VE: The consensus coding sequences of human breast and colorectal cancers. Science. 2006, 314: 268-274. 10.1126/science.1133427.CrossRefPubMed
17.
go back to reference Yang SH, Baek HA, Lee HJ, Park HS, Jang KY, Kang MJ, Lee DG, Lee YC, Moon WS, Chung MJ: Discoidin domain receptor 1 is associated with poor prognosis of non-small cell lung carcinomas. Oncol Rep. 2010, 24: 311-319.PubMed Yang SH, Baek HA, Lee HJ, Park HS, Jang KY, Kang MJ, Lee DG, Lee YC, Moon WS, Chung MJ: Discoidin domain receptor 1 is associated with poor prognosis of non-small cell lung carcinomas. Oncol Rep. 2010, 24: 311-319.PubMed
18.
go back to reference Zhang L, Wang S, Wang T, Wang Y, Wang J, Jiang L, Li S, Hu X, Wang Q: Upregulation of GRP78 and GRP94 and its function in chemotherapy resistance to VP-16 in human lung cancer cell line SK-MES-1. Cancer Invest. 2009, 27: 453-458. 10.1080/07357900802527239.CrossRefPubMed Zhang L, Wang S, Wang T, Wang Y, Wang J, Jiang L, Li S, Hu X, Wang Q: Upregulation of GRP78 and GRP94 and its function in chemotherapy resistance to VP-16 in human lung cancer cell line SK-MES-1. Cancer Invest. 2009, 27: 453-458. 10.1080/07357900802527239.CrossRefPubMed
19.
go back to reference Anttonen A, Heikkilä P, Kajanti M, Jalkanen M, Joensuu H: High syndecan-1 expression is associated with favourable outcome in squamous cell lung carcinoma treated with radical surgery. Lung Cancer. 2001, 32: 297-305. 10.1016/S0169-5002(00)00230-0.CrossRefPubMed Anttonen A, Heikkilä P, Kajanti M, Jalkanen M, Joensuu H: High syndecan-1 expression is associated with favourable outcome in squamous cell lung carcinoma treated with radical surgery. Lung Cancer. 2001, 32: 297-305. 10.1016/S0169-5002(00)00230-0.CrossRefPubMed
20.
go back to reference Qiu J, Choi G, Li L, Wang H, Pitteri SJ, Pereira-Faca SR, Krasnoselsky AL, Randolph TW, Omenn GS, Edelstein C, Barnett MJ, Thornquist MD, Goodman GE, Brenner DE, Feng Z, Hanash SM: Occurrence of autoantibodies to annexin I, 14-3-3 theta and LAMR1 in prediagnostic lung cancer sera. J Clin Oncol. 2008, 26: 5060-5066. 10.1200/JCO.2008.16.2388.CrossRefPubMedPubMedCentral Qiu J, Choi G, Li L, Wang H, Pitteri SJ, Pereira-Faca SR, Krasnoselsky AL, Randolph TW, Omenn GS, Edelstein C, Barnett MJ, Thornquist MD, Goodman GE, Brenner DE, Feng Z, Hanash SM: Occurrence of autoantibodies to annexin I, 14-3-3 theta and LAMR1 in prediagnostic lung cancer sera. J Clin Oncol. 2008, 26: 5060-5066. 10.1200/JCO.2008.16.2388.CrossRefPubMedPubMedCentral
21.
go back to reference Zachara BA, Marchaluk-Wiśniewska E, Maciag A, Pepliński J, Skokowski J, Lambrecht W: Decreased selenium concentration and glutathione peroxidase activity in blood and increase of these parameters in malignant tissue of lung cancer patients. Lung. 1997, 175: 321-332. 10.1007/PL00007578.CrossRefPubMed Zachara BA, Marchaluk-Wiśniewska E, Maciag A, Pepliński J, Skokowski J, Lambrecht W: Decreased selenium concentration and glutathione peroxidase activity in blood and increase of these parameters in malignant tissue of lung cancer patients. Lung. 1997, 175: 321-332. 10.1007/PL00007578.CrossRefPubMed
22.
go back to reference Mashkova TD, Oparina NI, Zinov’eva OL, Kropotova ES, Dubovaia VI, Poltaraus AB, Fridman MV, Kopantsev EP, Vinogradova TV, Zinov’eva MV, Laktionov KK, Kasymova OT, Zborovskaia IB, Sverdlov ED, Kiselev LL: Transcription TIMP3, DAPk1 and AKR1B10 genes in squamous cell lung cancer. Mol Biol (Mosk). 2006, 40: 1047-1054.CrossRef Mashkova TD, Oparina NI, Zinov’eva OL, Kropotova ES, Dubovaia VI, Poltaraus AB, Fridman MV, Kopantsev EP, Vinogradova TV, Zinov’eva MV, Laktionov KK, Kasymova OT, Zborovskaia IB, Sverdlov ED, Kiselev LL: Transcription TIMP3, DAPk1 and AKR1B10 genes in squamous cell lung cancer. Mol Biol (Mosk). 2006, 40: 1047-1054.CrossRef
23.
go back to reference Mansilla F, da Costa KA, Wang S, Kruhøffer M, Lewin TM, Orntoft TF, Coleman RA, Birkenkamp-Demtröder K: Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer. J Mol Med. 2009, 87: 85-97. 10.1007/s00109-008-0409-0.CrossRefPubMed Mansilla F, da Costa KA, Wang S, Kruhøffer M, Lewin TM, Orntoft TF, Coleman RA, Birkenkamp-Demtröder K: Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer. J Mol Med. 2009, 87: 85-97. 10.1007/s00109-008-0409-0.CrossRefPubMed
24.
go back to reference Otte S, Barlowe C: The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER. EMBO J. 2002, 21: 6095-6104. 10.1093/emboj/cdf598.CrossRefPubMedPubMedCentral Otte S, Barlowe C: The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER. EMBO J. 2002, 21: 6095-6104. 10.1093/emboj/cdf598.CrossRefPubMedPubMedCentral
25.
go back to reference Breuza L, Halbeisen R, Jenö P, Otte S, Barlowe C, Hong W, Hauri HP: Proteomics of endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes from brefeldin A-treated HepG2 cells identifies ERGIC-32, a new cycling protein that interacts with human Erv46. J Biol Chem. 2004, 279: 47242-47253. 10.1074/jbc.M406644200.CrossRefPubMed Breuza L, Halbeisen R, Jenö P, Otte S, Barlowe C, Hong W, Hauri HP: Proteomics of endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes from brefeldin A-treated HepG2 cells identifies ERGIC-32, a new cycling protein that interacts with human Erv46. J Biol Chem. 2004, 279: 47242-47253. 10.1074/jbc.M406644200.CrossRefPubMed
26.
go back to reference Welsh LM, Tong AH, Boone C, Jensen ON, Otte S: Genetic and molecular interactions of the Erv41p-Erv46p complex involved in transport between the endoplasmic reticulum and Golgi complex. J Cell Sci. 2006, 119: 4730-4740. 10.1242/jcs.03250.CrossRefPubMed Welsh LM, Tong AH, Boone C, Jensen ON, Otte S: Genetic and molecular interactions of the Erv41p-Erv46p complex involved in transport between the endoplasmic reticulum and Golgi complex. J Cell Sci. 2006, 119: 4730-4740. 10.1242/jcs.03250.CrossRefPubMed
27.
go back to reference Kitazume S, Tachida Y, Oka R, Shirotani K, Saido TC, Hashimoto Y: Alzheimer’s beta-secretase, beta-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase. Proc Natl Acad Sci USA. 2001, 98: 13554-13559. 10.1073/pnas.241509198.CrossRefPubMedPubMedCentral Kitazume S, Tachida Y, Oka R, Shirotani K, Saido TC, Hashimoto Y: Alzheimer’s beta-secretase, beta-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase. Proc Natl Acad Sci USA. 2001, 98: 13554-13559. 10.1073/pnas.241509198.CrossRefPubMedPubMedCentral
28.
go back to reference Nishikawa M, Kira Y, Yabunaka Y, Inoue M: Identification and characterization of endoplasmic reticulum-associated protein, ERp43. Gene. 2007, 386: 42-51. 10.1016/j.gene.2006.06.030.CrossRefPubMed Nishikawa M, Kira Y, Yabunaka Y, Inoue M: Identification and characterization of endoplasmic reticulum-associated protein, ERp43. Gene. 2007, 386: 42-51. 10.1016/j.gene.2006.06.030.CrossRefPubMed
29.
go back to reference Otte S, Belden WJ, Heidtman M, Liu J, Jensen ON, Barlowe C: Erv41p And Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex. J Cell Biol. 2001, 152: 503-518. 10.1083/jcb.152.3.503.CrossRefPubMedPubMedCentral Otte S, Belden WJ, Heidtman M, Liu J, Jensen ON, Barlowe C: Erv41p And Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex. J Cell Biol. 2001, 152: 503-518. 10.1083/jcb.152.3.503.CrossRefPubMedPubMedCentral
Metadata
Title
Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3as a novel lung cancer-related gene
Authors
Mingsong Wu
Tao Tu
Yunchao Huang
Yi Cao
Publication date
01-12-2013
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2013
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/1471-2407-13-44

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