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| Status |
Public on Sep 19, 2009 |
| Title |
NMD inhibition fails to identify tumour suppressor genes in microsatellite stable gastric cancer cell lines |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
Genome variation profiling by array
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| Summary |
Gastric cancers frequently show chromosomal alterations which can cause activation of oncogenes, and/or inactivation of tumour suppressor genes. In gastric cancer several chromosomal regions are described to be frequently lost, but for most of the regions, no tumour suppressor genes have been identified yet. The present study aimed to identify tumour suppressor genes inactivated by nonsense mutation and deletion in gastric cancer by means of GINI (gene identification by nonsense mediated decay inhibition) and whole genome copy number analysis.
Material and Methods: Two non-commercial gastric cancer cell lines, GP202 and IPA220, were transfected with siRNA directed against UPF1, to specifically inhibit the nonsense mediated decay (NMD) pathway, and with siRNA directed against non-specific siRNA duplexes (CVII) as a control. Microarray expression experiments were performed in triplicate on 4x44 K agilent arrays by hybridizing RNA from UPF1-transfected cells against non-specific CVII-transfected cells. In addition, array CGH of the two cell lines was performed on 4x44K agilent arrays to obtain the DNA copy number profiles. Mutation analysis of GINI candidates was performed by sequencing.
Results: UPF1 expression was reduced for >70% and >80% in the GP202 and IPA220 gastric cancer cell lines, respectively. Integration of array CGH and microarray expression data provided a list of 134 and 50 candidate genes inactivated by nonsense mutation and deletion for GP202 and IPA220, respectively. We selected 12 candidate genes for mutation analysis. Sequence analysis was performed on 11 genes. One gene, PLA2G4A, showed a silent mutation, and in two genes, CTSA and PTPRJ, missense mutations were detected. No nonsense mutations were detected in any of the 11 genes tested.
Conclusions: Although UPF1 was substantially repressed, thus resulting in the inhibition of the NMD system, we did not find genes inactivated by nonsense mutations. Our results show that the GINI strategy leads to a high number of false positives.
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| Overall design |
Two non-commercial gastric cancer cell lines, GP202 and IPA220, were transfected with siRNA directed against UPF1, to specifically inhibit the nonsense mediated decay (NMD) pathway, and with siRNA directed against non-specific siRNA duplexes (CVII) as a control. Microarray expression experiments were performed in triplicate on 4x44 K agilent arrays by hybridizing RNA from UPF1-transfected cells against non-specific CVII-transfected cells. In addition, array CGH of the two cell lines was performed on 4x44K agilent arrays to obtain the DNA copy number profiles. Mutation analysis of GINI candidates was performed by sequencing.
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| Contributor(s) |
Buffart TE, Tijssen M, El-Bchiri J, Duval A, van der Wal MA, Ylstra B, Seruca R, Meijer GA, Carvalho B |
| Citation(s) |
19563644 |
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| Submission date |
Sep 25, 2008 |
| Last update date |
Feb 22, 2018 |
| Contact name |
Daoud Sie |
| E-mail(s) |
d.sie@vumc.nl
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| Phone |
+31 20 4442428
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| Organization name |
Vrije Universiteit Medical Center
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| Department |
Pathology
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| Lab |
Microarray Core Facility
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| Street address |
De Boelelaan 1117
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| City |
Amsterdam |
| ZIP/Postal code |
1081 HV |
| Country |
Netherlands |
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| Platforms (2) |
| GPL4133 |
Agilent-014850 Whole Human Genome Microarray 4x44K G4112F (Feature Number version) |
| GPL5477 |
Agilent-014950 Human Genome CGH Microarray 4x44K (Feature Number version) |
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| Samples (14)
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| Relations |
| BioProject |
PRJNA110847 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE12928_RAW.tar |
181.2 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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