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Methylation analysis of promoter regions for selected tumour suppressor genes in DNA from white blood cells.

ABSTRACT: Methylation analysis of promoter regions for selected tumour suppressor genes in DNA from white blood cells.

PROVIDER: PRJNA832682 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18937246_1.fastq.gz	Fastqsanger.gz
	SRR18937246_2.fastq.gz	Fastqsanger.gz
	SRR18937247_1.fastq.gz	Fastqsanger.gz
	SRR18937247_2.fastq.gz	Fastqsanger.gz
	SRR18937248_1.fastq.gz	Fastqsanger.gz

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Project description: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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Methylation analysis of promoter regions for selected tumour suppressor genes in DNA from white blood cells.

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